Soybean event DP-305423-1 and compositions and methods for the identification and/or detection thereor

ABSTRACT

Compositions and methods related to transgenic high oleic acid/ALS inhibitor-tolerant soybean plants are provided. Specifically, the present invention provides soybean plants having a DP-305423-1 event which imparts a high oleic acid phenotype and tolerance to at least one ALS-inhibiting herbicide. The soybean plant harboring the DP-305423-1 event comprises genomic/transgene junctions having at least the polynucleotide sequence of SEQ ID NO:8, 9, 14, 15, 20, 21, 83 or 84. The characterization of the genomic insertion site of the DP-305423-1 event provides for an enhanced breeding efficiency and enables the use of molecular markers to track the transgene insert in the breeding populations and progeny thereof. Various methods and compositions for the identification, detection, and use of the soybean DP-305423-1 events are provided.

This application is a continuation of U.S. patent application Ser. No.11/927,884, filed Oct. 30, 2007, now U.S. Pat. No. 8,609,935, whichclaims the benefit of U.S. Provisional Application No. 60/863,721, filedOct. 31, 2006, and U.S. Provisional Application No. 60/942,676, filedJun. 8, 2007, the entire contents of each are herein incorporated byreference.

FIELD OF THE INVENTION

This invention is in the field of molecular biology. More specifically,this invention pertains to plants that display both a high oleic acidphenotype and a herbicide tolerance phenotype conferred by suppressionof a FAD2 gene in conjunction with the expression of a sequence thatconfers tolerance to inhibitors of ALS.

BACKGROUND OF THE INVENTION

The expression of foreign genes in plants is known to be influenced bytheir location in the plant genome, perhaps due to chromatin structure(e.g., heterochromatin) or the proximity of transcriptional regulatoryelements (e.g., enhancers) close to the integration site (Weising et al.(1988) Ann. Rev. Genet 22: 421-477). At the same time the presence ofthe transgene at different locations in the genome influences theoverall phenotype of the plant in different ways. For this reason, it isoften necessary to screen a large number of events in order to identifyan event characterized by optimal expression of an introduced gene ofinterest. For example, it has been observed in plants and in otherorganisms that there may be a wide variation in levels of expression ofan introduced gene among events. There may also be differences inspatial or temporal patterns of expression, for example, differences inthe relative expression of a transgene in various plant tissues, thatmay not correspond to the patterns expected from transcriptionalregulatory elements present in the introduced gene construct. It is alsoobserved that the transgene insertion can affect the endogenous geneexpression. For these reasons, it is common to produce hundreds tothousands of different events and screen those events for a single eventthat has desired transgene expression levels and patterns for commercialpurposes. An event that has desired levels or patterns of transgeneexpression is useful for introgressing the transgene into other geneticbackgrounds by sexual outcrossing using conventional breeding methods.Progeny of such crosses maintain the transgene expressioncharacteristics of the original transformant. This strategy is used toensure reliable gene expression in a number of varieties that are welladapted to local growing conditions.

It would be advantageous to be able to detect the presence of aparticular event in order to determine whether progeny of a sexual crosscontain a transgene of interest. In addition, a method for detecting aparticular event would be helpful for complying with regulationsrequiring the pre-market approval and labeling of foods derived fromrecombinant crop plants, or for use in environmental monitoring,monitoring traits in crops in the field, or monitoring products derivedfrom a crop harvest, as well as, for use in ensuring compliance ofparties subject to regulatory or contractual terms.

In the commercial production of crops, it is desirable to easily andquickly eliminate unwanted plants (i.e., “weeds”) from a field of cropplants. An ideal treatment would be one which could be applied to anentire field but which would eliminate only the unwanted plants whileleaving the crop plants unharmed. One such treatment system wouldinvolve the use of crop plants which are tolerant to a herbicide so thatwhen the herbicide was sprayed on a field of herbicide-tolerant cropplants, the crop plants would continue to thrive whilenon-herbicide-tolerant weeds were killed or severely damaged.

Plant lipids find their major use as edible oils in the form oftriacylglycerols. The specific performance and health attributes ofedible oils are determined largely by their fatty acid composition. Mostvegetable oils derived from commercial plant varieties are composedprimarily of palmitic (16:0), stearic (18:0), oleic (18:1), linoleic(18:2) and linolenic (18:3) acids. Palmitic and stearic acids are,respectively, 16- and 18-carbon-long, saturated fatty acids. Oleic,linoleic, and linolenic acids are 18-carbon-long, unsaturated fattyacids containing one, two, and three double bonds, respectively. Oleicacid is referred to as a mono-unsaturated fatty acid, while linoleic andlinolenic acids are referred to as poly-unsaturated fatty acids.

A vegetable oil low in total saturates and high in mono-unsaturateswould provide significant health benefits to consumers as well aseconomic benefits to oil processors. As an example, canola oil isconsidered a very healthy oil. However, in use, the high level ofpoly-unsaturated fatty acids in canola oil renders the oil unstable,easily oxidized, and susceptible to development of disagreeable odorsand flavors (Gailliard, 1980, Vol. 4, pp. 85-116 In: Stumpf, P. K., Ed.,The Biochemistry of Plants, Academic Press, New York). The levels ofpoly-unsaturates may be reduced by hydrogenation, but the expense ofthis process and the concomitant production of nutritionallyquestionable trans isomers of the remaining unsaturated fatty acidsreduces the overall desirability of the hydrogenated oil (Mensink etal., New England J. Medicine (1990) N323: 439-445). Similar problemsexist with soybean and corn oils.

SUMMARY OF THE INVENTION

Compositions and methods related to transgenic high oleic acid/ALSinhibitor-tolerant soybean plants are provided. Specifically, thepresent invention provides soybean plants containing a DP-305423-1 eventwhich imparts a high oleic acid phenotype and tolerance to at least oneALS-inhibiting herbicide. The soybean plant harboring the DP-305423-1event at the recited chromosomal location comprises genomic/transgenejunctions having at least the polynucleotide sequence of SEQ ID NO:8, 9,14, 15, 20, 21, 83 or 84. The characterization of the genomic insertionsite of the DP-305423-1 event provides for an enhanced breedingefficiency and enables the use of molecular markers to track thetransgene insert in the breeding populations and progeny thereof.Various methods and compositions for the identification, detection, anduse of the soybean DP-305423-1 event are provided.

In one embodiment, the present invention includes an isolatedpolynucleotide comprising SEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82, 83, 84, 85, 86, 87 or88.

In another embodiment, the present invention includes a soybean plant ora soybean seed comprising SEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82, 83, 84, 85, 86, 87 or88.

In another embodiment, the present invention includes a method foridentifying a biological sample comprising: a) contacting saidbiological sample with a first and a second primer; b) amplifying apolynucleotide comprising any of SEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82, 83, 84, 85, 86,87 or 88; and c) confirming said biological sample comprises apolynucleotide comprising any of SEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82, 83, 84, 85, 86,87 or 88. The method may further comprise detecting a polynucleotidecomprising SEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 82, 83, 84, 85, 86, 87 or 88 byhybridization to a probe, wherein said probe hybridizes under stringenthybridization conditions with a polynucleotide comprising SEQ ID NO:5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 82, 83, 84, 85, 86, 87 or 88. The first or second primer maycomprise a fragment of a 5′ genomic region, a 3′ genomic region or aninsert region of SEQ ID NO:5, 6, 7 or 82. The first or second primer maycomprise at least 8 consecutive nucleotides of a 5′ genomic region, a 3′genomic region or an insert region of SEQ ID NO:5, 6, 7 or 82. One ofthe first or second primers may comprise a fragment of a 5′ genomicregion of SEQ ID NO:5, 6, 7 or 82 and the other of the first or secondprimers may comprise a fragment of a 3′ genomic region of SEQ ID NO:5,6, 7 or 82. The first or second primer may comprise SEQ ID NO:26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,89, 90, 91, 92, 93 or 94.

In another embodiment, the present invention includes a method ofdetecting the presence of a polynucleotide comprising SEQ ID NO:5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,82, 83, 84, 85, 86, 87 or 88 in a biological sample comprising DNA,comprising: (a) extracting a DNA sample from said biological sample; (b)contacting said DNA sample with at least one pair of DNA primermolecules selected from the group consisting of: i) the sequencescomprising SEQ ID NO:26 and SEQ ID NO:27; ii) the sequences comprisingSEQ ID NO:29 and SEQ ID NO:30; iii) the sequences comprising SEQ IDNO:31 and SEQ ID NO:32; iv) the sequences comprising SEQ ID NO:33 andSEQ ID NO:34; v) the sequences comprising SEQ ID NO:35 and SEQ ID NO:36;vi) the sequences comprising SEQ ID NO:37 and SEQ ID NO:38; vii) thesequences comprising SEQ ID NO:39 and SEQ ID NO:40; viii) the sequencescomprising SEQ ID NO:41 and SEQ ID NO:42; ix) the sequences comprisingSEQ ID NO:43 and SEQ ID NO:44; x) the sequences comprising SEQ ID NO:45and SEQ ID NO:46; xi) the sequences comprising SEQ ID NO:47 and SEQ IDNO:48; xii) the sequences comprising SEQ ID NO:47 and SEQ ID NO:49;xiii) the sequences comprising SEQ ID NO:50 and SEQ ID NO:51; xiv) thesequences comprising SEQ ID NO:52 and SEQ ID NO:53; xv) the sequencescomprising SEQ ID NO:54 and SEQ ID NO:49; xvi) the sequences comprisingSEQ ID NO:55 and SEQ ID NO:46; xvii) the sequences comprising SEQ IDNO:33 and SEQ ID NO:56; xviii) the sequences comprising SEQ ID NO:57 andSEQ ID NO:58; xix) the sequences comprising SEQ ID NO:59 and SEQ IDNO:60; xx) the sequences comprising SEQ ID NO:61 and SEQ ID NO:36; xxi)the sequences comprising SEQ ID NO:35 and SEQ ID NO:62; xxii) thesequences comprising SEQ ID NO:37 and SEQ ID NO:63; xxiii) the sequencescomprising SEQ ID NO:64 and SEQ ID NO:65; xxiv) the sequences comprisingSEQ ID NO:66 and SEQ ID NO:67; xxv) the sequences comprising SEQ IDNO:68 and SEQ ID NO:69; xxvi) the sequences comprising SEQ ID NO:70 andSEQ ID NO:71; xxvii) the sequences comprising SEQ ID NO:72 and SEQ IDNO:73; xxviii) the sequences comprising SEQ ID NO:74 and SEQ ID NO:75;xxix) the sequences comprising SEQ ID NO:76 and SEQ ID NO:77; xxx) thesequences comprising SEQ ID NO:78 and SEQ ID NO:79; xxxi) the sequencescomprising SEQ ID NO:80 and SEQ ID NO:81; and xxxii) the sequencescomprising SEQ ID NO:89 and SEQ ID NO:90 (c) providing DNA amplificationreaction conditions; (d) performing said DNA amplification reaction,thereby producing a DNA amplicon molecule; and (e) detecting said DNAamplicon molecule, wherein the detection of said DNA amplicon moleculein said DNA amplification reaction indicates the presence of apolynucleotide comprising SEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82, 83, 84, 85, 86, 87 or88.

In another embodiment, the present invention includes a method ofdetecting the presence of SEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82, 83, 84, 85, 86, 87 or 88in a biological sample, the method comprising: (a) contacting thebiological sample comprising DNA under stringent hybridizationconditions with a polynucleotide probe wherein said probe hybridizesunder stringent hybridization conditions with a polynucleotidecomprising SEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 82, 83, 84, 85, 86, 87 or 88; (b) detectinghybridization of the probe to the DNA. The biological sample maycomprise soybean tissue.

In another embodiment, the present invention includes an isolated DNAprimer comprising at least one sequence selected from the groupconsisting of SEQ ID NO:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 89, 90, 91, 92, 93 or 94 or itscomplement.

In another embodiment, the present invention includes a pair of DNAprimers comprising a first DNA primer and a second DNA primer, whereinthe DNA primers are of a sufficient length of contiguous nucleotides ofSEQ ID NO:5, 6, 7 or 82, to function as DNA primers diagnostic of DNAcomprising SEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 82, 83, 84, 85, 86, 87 or 88.

In another embodiment, the present invention includes a DNA probewherein the DNA probe is of a sufficient length of contiguousnucleotides of SEQ ID NO:5, 6, 7 or 82, to function as a DNA probediagnostic of DNA comprising SEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82, 83, 84, 85, 86, 87or 88.

In another embodiment, the present invention includes a method forscreening seed for a polynucleotide comprising SEQ ID NO:5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82, 83,84, 85, 86, 87 or 88, comprising: a) contacting a sample comprising DNAfrom said seed with a first and a second DNA primer; b) amplifying apolynucleotide comprising a polynucleotide comprising SEQ ID NO:5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,82, 83, 84, 85, 86, 87 or 88; and c) detecting said amplifiedpolynucleotide.

In another embodiment, the present invention includes a method forscreening seed for the presence of a polynucleotide comprising SEQ IDNO:5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 82, 83, 84, 85, 86, 87 or 88 comprising: (a) contacting asample comprising DNA from said seed under stringent hybridizationconditions with a polynucleotide probe that hybridizes under stringenthybridization conditions with a polynucleotide comprising SEQ ID NO:5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 82, 83, 84, 85, 86, 87 or 88; and (b) detecting hybridization of theprobe to the DNA.

In another embodiment, the present invention includes a method ofproducing a high oleic acid and ALS inhibitor tolerant plant comprisingbreeding a plant comprising a polynucleotide comprising SEQ ID NO:5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,82, 83, 84, 85, 86, 87 or 88, and selecting progeny by analyzing forprogeny that comprise a polynucleotide comprising SEQ ID NO: 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82,83, 84, 85, 86, 87 or 88.

In another embodiment, the present invention includes an isolated DNAsequence comprising at least one nucleotide sequence selected from thegroup consisting of: (a) a nucleotide sequence set forth in SEQ IDNO:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 89, 90, 91, 92, 93 or 94; and (b) a full-length complementof the nucleotide sequence of (a).

In another embodiment, the present invention includes a pair of isolatedDNA primer sequences, each comprising at least ten nucleotides and whichwhen used together in a DNA amplification procedure will produce a DNAamplicon comprising a polynucleotide comprising SEQ ID NO:5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82, 83,84, 85, 86, 87 or 88. The pair of isolated DNA primer sequences maycomprise a first primer sequence chosen from the group consisting of: a)a 5′ genomic region of SEQ ID NO: 5, 6, 7 or 82; and b) a 3′ genomicregion of SEQ ID NO: 5, 6, 7 or 82; and a second primer sequence chosenfrom an insert region of SEQ ID NO: 5, 6, 7 or 82.

In another embodiment, the present invention includes a method forcontrolling weeds in an area of cultivation comprising applying aneffective amount of an ALS inhibitor to the area of cultivationcomprising soybean plants comprising a polynucleotide comprising SEQ IDNO:5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 82, 83, 84, 85, 86, 87 or 88. The ALS inhibitor may be asulfonylurea herbicide or an imidazolinone herbicide. A combination ofdifferent ALS inhibitors may be used. The ALS inhibitor or combinationof ALS inhibitors may be used in further combination with one or morenon-ALS inhibitor herbicides.

In another embodiment, the present invention includes a DNA expressionconstruct comprising the isolated polynucleotide of the inventionoperably linked to at least one regulatory sequence.

In another embodiment, the present invention includes transgenic progenyplants obtained from the transgenic seed of the invention.

In another embodiment, the present invention includes a recombinant DNAconstruct comprising: a first and second expression cassette, whereinsaid first expression cassette in operable linkage comprises: (a) asoybean KTi3 promoter; (b) a gm-fad2-1 fragment; and (c) a soybean KTi3transcriptional terminator; and said second expression cassettecomprises in operable linkage: (i) a soybean SAMS promoter; (ii) asoybean SAMS 5′ untranslated leader and intron; (iii) a soybean gm-hraencoding DNA molecule; and (iv) a soybean als transcriptionalterminator.

In another embodiment, the present invention includes a plant or seedcomprising the recombinant DNA construct of claim 27. The plant or seedmay be a soybean plant or a soybean seed.

BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCE LISTING

FIG. 1 provides a schematic map of fragment PHP19340A indicating variousgenetic elements and restriction enzyme sites for Nco I and Hind III.

FIG. 2 provides a schematic map of fragment PHP17752A indicating variousgenetic elements and restriction enzyme sites for Nco I and Hind III.

FIG. 3 provides a schematic map of expression vector PHP19340 indicatingvarious genetic elements and restriction enzyme sites for Asc I, Nco Iand Hind III.

FIG. 4 provides a schematic map of expression vector PHP17752 indicatingvarious genetic elements and restriction enzyme sites for Asc I, Nco Iand Hind III.

FIG. 5 shows a Southern hybridization experiment of genomic DNA fromsoybean leaf tissue of individual plants of DP-305423-1 (T5 and T4generation) and of unmodified control (Jack), digested with Hind III andprobed with the gm-fad2-1 gene probe.

FIG. 6 shows a Southern hybridization experiment of genomic DNA isolatedfrom soybean leaf tissue of individual plants of DP-305423-1 (T5 and T4generation) and of unmodified control (Jack), digested with Nco I andprobed with the gm-fad2-1 gene probe.

FIG. 7 shows a Southern hybridization experiment of genomic DNA isolatedfrom soybean leaf tissue of individual plants of DP-305423-1 (T5 and T4generation) and of unmodified control (Jack). digested with Hind III andprobed with the gm-hra gene probe.

FIG. 8 shows a Southern hybridization experiment of genomic DNA isolatedfrom soybean leaf tissue of individual plants of DP-305423-1 (T5 and T4generation) and of unmodified control (Jack), digested with Nco I andprobed with the gm-hra gene probe.

FIG. 9 provides a schematic map of Contig-1 indicating various geneticelements within Insertion-1.

FIG. 10 provides a schematic map of Contig-2 indicating various geneticelements within Insertion-2.

FIG. 11 provides a schematic map of Contig-3 indicating various geneticelements within Insertion-3.

FIG. 12 provides a schematic map of Contig-4 indicating various geneticelements within Insertion-4.

Table 1 presents a description of the following sequences that arepresent in the Sequence Listing: (1) the insert sequences used to createthe DP-305423-1 event and the vectors from which they are derived; (2)the genomic DNA sequences present in Contig-1, Contig-2, Contig-3 andContig-4; (3) the 5′ and 3′ junction sequences, at which transgenicinsert and endogenous soybean genomic sequence are joined, for each ofthe four contigs; and (4) primer sequences that can be used to amply 5′and 3′ junction sequences from each of the four contigs.

TABLE 1 Summary Table of SEQ ID NOS SEQ ID NO Description 1 PHP19340A 2PHP17752A 3 PHP19340 4 PHP17752 5 DP-305423-1 Contig-1 6 DP-305423-1Contig-2 7 DP-305423-1 Contig-3 8 Contig-1 20-nt 5′ junction (5′genomic/5′ transgene; 10-nt/10-nt) 9 Contig-1 20-nt 3′ junction (3′transgene/3′ genomic; 10-nt/10-nt) 10 Contig-1 40-nt 5′ junction (5′genomic/5′ transgene; 20-nt/20-nt) 11 Contig-1 40-nt 3′ junction (3′transgene/3′ genomic; 20-nt/20-nt) 12 Contig-1 60-nt 5′ junction (5′genomic/5′ transgene; 30-nt/30-nt) 13 Contig-1 60-nt 3′ junction (3′transgene/3′ genomic; 30-nt/30-nt) 14 Contig-2 20-nt 5′ junction (5′genomic/5′ transgene; 10-nt/10-nt) 15 Contig-2 20-nt 3′ junction (3′transgene/3′ genomic; 10-nt/10-nt) 16 Contig-2 40-nt 5′ junction (5′genomic/5′ transgene; 20-nt/20-nt) 17 Contig-2 40-nt 3′ junction (3′transgene/3′ genomic; 20-nt/20-nt) 18 Contig-2 60-nt 5′ junction (5′genomic/5′ transgene; 30-nt/30-nt) 19 Contig-2 60-nt 3′ junction (3′transgene/3′ genomic; 30-nt/30-nt) 20 Contig-3 20-nt 5′ junction (5′genomic/5′ transgene; 10-nt/10-nt) 21 Contig-3 20-nt 3′ junction (3′transgene/3′ genomic; 10-nt/10-nt) 22 Contig-3 40-nt 5′ junction (5′genomic/5′ transgene; 20-nt/20-nt) 23 Contig-3 40-nt 3′ junction (3′transgene/3′ genomic; 20-nt/20-nt) 24 Contig-3 60-nt 5′ junction (5′genomic/5′ transgene; 30-nt/30-nt) 25 Contig-3 60-nt 3′ junction (3′transgene/3′ genomic; 30-nt/30-nt) 26 05-O-975 Contig-1 5′ junctionforward primer 27 05-O-977 Contig-1 5′ junction reverse primer 2805-QP22 Contig-1 5′ junction probe 29 06-O-1573 Contig-1 5′ junctionforward primer 30 06-O-1487 Contig-1 5′ junction reverse primer 3106-O-1414 Contig-1 3′ junction forward primer 32 06-O-1579 Contig-1 3′junction reverse primer 33 06-O-1577 Contig-1 3′ junction forward primer34 06-O-1579 Contig-1 3′ junction reverse primer 35 06-O-1586 Contig-25′ junction forward primer 36 06-O-1585 Contig-2 5′ junction reverseprimer 37 06-O-1404 Contig-2 3′ junction forward primer 38 06-O-1590Contig-2 3′ junction reverse primer 39 06-O-1626 Contig-3 5′ junctionforward primer 40 06-O-1366 Contig-3 5′ junction reverse primer 4106-O-1569 Contig-3 3′ junction forward primer 42 06-O-1551 Contig-3 3′junction reverse primer 43 06-O-1571 Contig-1 5′ junction forward primer44 06-O-1572 Contig-1 5′ junction reverse primer 45 06-O-1351 Contig-15′ junction forward primer 46 06-O-1367 Contig-1 5′ junction reverseprimer 47 06-O-1357 Contig-1 insert forward primer 48 06-O-1368 Contig-1insert reverse primer 49 06-O-1369 Contig-1 insert reverse primer 5006-O-1356 Contig-1 insert forward primer 51 06-O-1371 Contig-1 insertreverse primer 52 06-O-1360 Contig-1 insert forward primer 53 06-O-1423Contig-1 insert reverse primer 54 06-O-1363 Contig-1 insert forwardprimer 55 06-O-1421 Contig-1 insert forward primer 56 06-O-1578 Contig-13′ junction reverse primer 57 07-O-1889 Contig-1 5′ region forwardprimer 58 07-O-1940 Contig-1 5′ region reverse primer 59 07-O-1892Contig-1 3′ region reverse primer 60 07-O-1894 Contig-1 3′ regionforward primer 61 06-O-1588 Contig-2 5′ junction forward primer 6206-O-1403 Contig-2 5′ junction reverse primer 63 06-O-1592 Contig-2 3′junction reverse primer 64 07-O-1895 Contig-2 5′ region forward primer65 07-O-1898 Contig-2 5′ region reverse primer 66 07-O-1905 Contig-2 3′region forward primer 67 07-O-1903 Contig-2 3′ region reverse primer 6806-O-1669 Contig-3 5′ junction forward primer 69 06-O-1426 Contig-3 5′junction reverse primer 70 06-O-1355 Contig-3 insert forward primer 7106-O-1459 Contig-3 insert reverse primer 72 05-O-1182 Contig-3 3′junction forward primer 73 06-O-1672 Contig-3 3′ junction reverse primer74 07-O-1881 Contig-3 5′ region forward primer 75 07-O-1882 Contig-3 5′region reverse primer 76 07-O-1886 Contig-3 3′ region forward primer 7707-O-1884 Contig-3 3′ region reverse primer 78 HOS-A Contig-4 5′junction forward primer 79 HOS-B Contig-4 5′ junction reverse primer 80HOS-C Contig-4 3′ junction reverse primer 81 HOS-D Contig-4 3′ junctionforward primer 82 DP-305423-1 Contig-4 83 Contig-4 20-nt 5′ junction (5′genomic/5′ transgene; 10-nt/10-nt) 84 Contig-4 20-nt 3′ junction (3′transgene/3′ genomic; 10-nt/10-nt) 85 Contig-4 40-nt 5′ junction (5′genomic/5′ transgene; 20-nt/20-nt) 86 Contig-4 40-nt 3′ junction (3′transgene/3′ genomic; 20-nt/20-nt) 87 Contig-4 60-nt 5′ junction (5′genomic/5′ transgene; 30-nt/30-nt) 88 Contig-4 60-nt 3′ junction (3′transgene/3′ genomic; 30-nt/30-nt) 89 Contig-1 5′ junction QPCR forwardprimer 90 Contig-1 5′ junction QPCR reverse primer 91 Contig-1 5′junction QPCR probe 92 SAMS-HRA QPCR forward primer 93 SAMS-HRA QPCRreverse primer 94 SAMS-HRA QPCR probe

DETAILED DESCRIPTION OF THE INVENTION

The present inventions now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the inventions are shown. Indeed, these inventions may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

The following abbreviations are used in describing the presentinvention.

-   -   ALS acetolactate synthase protein    -   bp base pair    -   FAD2 microsomal omega-6 desaturase protein    -   gm-fad2-1 soybean microsomal omega-6 desaturase gene 1    -   gm-als wild type acetolactate synthase gene from soybean    -   gm-hra modified version of acetolactate synthase gene from        soybean    -   kb kilobase    -   PCR polymerase chain reaction    -   UTR untranslated region

Compositions and methods related to transgenic high oleic acid/ALSinhibitor-tolerant soybean plants are provided. Specifically, thepresent invention provides soybean plants having event DP-305423-1. Asoybean plant having “event DP-305423-1” has been modified by theinsertion of a suppression cassette containing a 597 bp fragment of thesoybean microsomal omega-6 desaturase gene 1 (gm-fad2-1) and anexpression cassette containing a modified version of the soybeanacetolactate synthase gene (gm-hra). The insertion of the gm-fad2-1suppression cassette in the plant confers a high oleic acid phenotype.The insertion of the gm-hra gene produces a modified form of theacetolactate synthase (ALS) enzyme. ALS is essential for branched chainamino acid biosynthesis and is inhibited by certain herbicides. Themodification in the gm-hra gene overcomes this inhibition and thusprovides tolerance to a wide range of ALS-inhibiting herbicides. Thus, asoybean plant having a DP-305423-1 event has a high oleic acid phenotypeand is tolerant at least one ALS-inhibiting herbicide.

The polynucleotides conferring the high oleic acid phenotype and ALSinhibitor tolerance are genetically linked in the soybean genome in theDP-305423-1 soybean event. The soybean plant harboring the DP-305423-1event comprises genomic/transgene junctions having at least thepolynucleotide sequence of SEQ ID NO: 8, 9, 14, 15, 20, 21, 83, and 84.The characterization of the genomic insertion site of the DP-305423-1event provides for an enhanced breeding efficiency and enables the useof molecular markers to track the transgene insert in the breedingpopulations and progeny thereof. Various methods and compositions forthe identification, detection, and use of the soybean DP-305423-1 eventsare provided herein. As used herein, the term “event DP-305423-1specific” refers to a polynucleotide sequence which is suitable fordiscriminatively identifying event DP-305423-1 in plants, plantmaterial, or in products such as, but not limited to, food or feedproducts (fresh or processed) comprising, or derived from plantmaterial.

As used herein, the term “soybean” means Glycine max and includes allplant varieties that can be bred with soybean. As used herein, the termplant includes plant cells, plant organs, plant protoplasts, plant celltissue cultures from which plants can be regenerated, plant calli, plantclumps, and plant cells that are intact in plants or parts of plantssuch as embryos, pollen, ovules, seeds, leaves, flowers, branches,fruit, stalks, roots, root tips, anthers, and the like. Grain isintended to mean the mature seed produced by commercial growers forpurposes other than growing or reproducing the species. Progeny,variants, and mutants of the regenerated plants are also included withinthe scope of the invention, provided that these parts comprise aDP-305423-1 event.

A transgenic “event” is produced by transformation of plant cells with aheterologous DNA construct(s), including a nucleic acid expressioncassette that comprises a transgene of interest, the regeneration of apopulation of plants resulting from the insertion of the transgene intothe genome of the plant, and selection of a particular plantcharacterized by insertion into a particular genome location. An eventis characterized phenotypically by the expression of the transgene(s).At the genetic level, an event is part of the genetic makeup of a plant.The term “event” also refers to progeny produced by a sexual outcrossbetween the transformant and another variety that include theheterologous DNA. Even after repeated back-crossing to a recurrentparent, the inserted DNA and flanking DNA from the transformed parent ispresent in the progeny of the cross at the same chromosomal location.The term “event” also refers to DNA from the original transformantcomprising the inserted DNA and flanking sequence immediately adjacentto the inserted DNA that would be expected to be transferred to aprogeny that receives inserted DNA including the transgene of interestas the result of a sexual cross of one parental line that includes theinserted DNA (e.g., the original transformant and progeny resulting fromselfing) and a parental line that does not contain the inserted DNA.

As used herein, “insert DNA” refers to the heterologous DNA within theexpression cassettes used to transform the plant material while“flanking DNA” can comprise either genomic DNA naturally present in anorganism such as a plant, or foreign (heterologous) DNA introduced viathe transformation process which is extraneous to the original insertDNA molecule, e.g. fragments associated with the transformation event. A“flanking region” or “flanking sequence” as used herein refers to asequence of at least 20, 50, 100, 200, 300, 400, 1000, 1500, 2000, 2500,or 5000 base pair or greater which is located either immediatelyupstream of and contiguous with or immediately downstream of andcontiguous with the original foreign insert DNA molecule. Non-limitingexamples of the flanking regions of the DP-305423-1 event are set forthin SEQ ID NO:5, 6, 7 and 82, and variants and fragments thereof.

Transformation procedures leading to random integration of the foreignDNA will result in transformants containing different flanking regionscharacteristic of and unique for each transformant. A “junction” is apoint where two specific DNA fragments join. For example, a junctionexists where insert DNA joins flanking genomic DNA. A junction pointalso exists in a transformed organism where two DNA fragments jointogether in a manner that is modified from that found in the nativeorganism. As used herein, “junction DNA” refers to DNA that comprises ajunction point. Non-limiting examples of junction DNA from theDP-305423-1 event set are forth in SEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82, 83, 84, 85, 86,87 or 88, or variants and fragments thereof.

A DP-305423-1 plant can be bred by first sexually crossing a firstparental soybean plant grown from the transgenic DP-305423-1 soybeanplant (or progeny thereof derived from transformation with theexpression cassettes of the embodiments of the present invention thatconfer herbicide tolerance) and a second parental soybean plant thatlacks the herbicide tolerance phenotype, thereby producing a pluralityof first progeny plants; and then selecting a first progeny plant thatdisplays the desired herbicide tolerance; and selfing the first progenyplant, thereby producing a plurality of second progeny plants; and thenselecting from the second progeny plants which display the desiredherbicide tolerance. These steps can further include the back-crossingof the first herbicide tolerant progeny plant or the second herbicidetolerant progeny plant to the second parental soybean plant or a thirdparental soybean plant, thereby producing a soybean plant that displaysthe desired herbicide tolerance. It is further recognized that assayingprogeny for phenotype is not required. Various methods and compositions,as disclosed elsewhere herein, can be used to detect and/or identify theDP-305423-1 event.

It is also to be understood that two different transgenic plants canalso be mated to produce offspring that contain two independentlysegregating added, exogenous genes. Selfing of appropriate progeny canproduce plants that are homozygous for both added, exogenous genes.Back-crossing to a parental plant and out-crossing with a non-transgenicplant are also contemplated, as is vegetative propagation. Descriptionsof other breeding methods that are commonly used for different traitsand crops can be found in one of several references, e.g., Fehr, inBreeding Methods for Cultivar Development, Wilcos J. ed., AmericanSociety of Agronomy, Madison Wis. (1987).

One particularly useful application of the claimed invention is tocombine the high oleic acid trait of the DP-305423-1 event with othersoybean lines that have altered fatty acid compositions to obtainprogeny lines with novel fatty acid compositions and/or improvedagronomic traits. The other soybean lines may be mutant lines,transgenic lines, or transgenic lines that also comprise a mutated gene.The transgenes of DP-305423-1 may be combined with mutant genes or othertransgenes either by making a genetic cross or by transforming the othersoybean line with the recombinant DNA constructs of the invention.

As examples, the high oleic acid trait of the invention can be combinedwith a mutant line having a high stearic acid phenotype, such as soybeanline A6 [Hammond, E. G. and Fehr, W. R. (1983)] or with a mutant linehaving a low linolenic acid phenotype such as soybean mutant lines A5,A23, A16 and C1640 [Fehr, W. R. et al. (1992) in Crop Science32:903-906]. Oils produced from such combinations would provide improvedfeedstocks for production of margarines, shortenings, spray coating andfrying oils and would eliminate or reduce the need for hydrogenation.Furthermore, these oils would provide a health benefit for consumers,for example by reducing or eliminating trans fatty acids which have beenfound to be associated with high risk to cardiovascular diseases.

The high oleic acid trait of the invention also can be combined withmutant lines that have a high oleic acid phenotype. Examples of higholeic acid mutant lines include soybean lines A5 and N782245 [Martin, B.A. and Rinne, R. W. (1985) Crop Science 25:1055-1058].

As used herein, the use of the term “polynucleotide” is not intended tolimit the present invention to polynucleotides comprising DNA. Those ofordinary skill in the art will recognize that polynucleotides, cancomprise ribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thepolynucleotides of the invention also encompass all forms of sequencesincluding, but not limited to, single-stranded forms, double-strandedforms, hairpins, stem-and-loop structures, and the like.

A DP-305423-1 plant comprises a suppression cassette containing a 597 bpfragment of the soybean microsomal omega-6 desaturase gene 1 (gm-fad2-1)and an expression cassette containing a modified version of the soybeanacetolactate synthase gene (gm-hra). The cassette can include 5′ and 3′regulatory sequences operably linked to the gm-fad2-1 and the gm-hrapolynucleotides. “Operably linked” is intended to mean a functionallinkage between two or more elements. For example, an operable linkagebetween a polynucleotide of interest and a regulatory sequence (i.e., apromoter) is functional link that allows for the expression of thepolynucleotide of interest. Operably linked elements may be contiguousor non-contiguous. When used to refer to the joining of two proteincoding regions, by operably linked it is intended that the codingregions are in the same reading frame. The cassette may additionallycontain at least one additional gene to be cotransformed into theorganism. Alternatively, the additional gene(s) can be provided onmultiple expression cassettes. Such an expression cassette is providedwith a plurality of restriction sites and/or recombination sites forinsertion of the polynucleotide to be under the transcriptionalregulation of the regulatory regions. The expression cassette mayadditionally contain selectable marker genes.

The expression cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region(i.e., a promoter), a coding region, and a transcriptional andtranslational termination region functional in plants. “Promoter” refersto a nucleotide sequence capable of controlling the expression of acoding sequence or functional RNA. In general, a coding sequence islocated 3′ to a promoter sequence. The promoter sequence can compriseproximal and more distal upstream elements, the latter elements areoften referred to as enhancers. Accordingly, an “enhancer” is anucleotide sequence that can stimulate promoter activity and may be aninnate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter. Promoters may bederived in their entirety from a native gene, or be composed ofdifferent elements derived from different promoters found in nature, oreven comprise synthetic nucleotide segments. It is understood by thoseskilled in the art that different promoters may direct the expression ofa gene in different tissues or cell types, or at different stages ofdevelopment, or in response to different environmental conditions.Promoters that cause a nucleic acid fragment to be expressed in mostcell types at most times are commonly referred to as “constitutivepromoters”. New promoters of various types useful in plant cells areconstantly being discovered; numerous examples may be found in thecompilation by Okamuro and Goldberg (1989) Biochemistry of Plants 15:1-82. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined,nucleic acid fragments of different lengths may have identical promoteractivity.

The expression cassettes may also contain 5′ leader sequences. Suchleader sequences can act to enhance translation. The regulatory regions(i.e., promoters, transcriptional regulatory regions, RNA processing orstability regions, introns, polyadenylation signals, and translationaltermination regions) and/or the coding region may be native/analogous orheterologous to the host cell or to each other.

The “translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect numerous parameters including, processing of theprimary transcript to mRNA, mRNA stability and/or translationefficiency. Examples of translation leader sequences have been described(Turner and Foster (1995) Mol. Biotechnol. 3: 225-236). The “3′non-coding sequences” refer to nucleotide sequences located downstreamof a coding sequence and include polyadenylation recognition sequencesand other sequences encoding regulatory signals capable of affectingmRNA processing or gene expression. The polyadenylation signal isusually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. (1989) PlantCell 1: 671-680.

As used herein, “heterologous” in reference to a sequence is a sequencethat originates from a foreign species, or, if from the same species, issubstantially modified from its native form in composition and/orgenomic locus by deliberate human intervention. For example, a promoteroperably linked to a heterologous polynucleotide is from a speciesdifferent from the species from which the polynucleotide was derived,or, if from the same/analogous species, one or both are substantiallymodified from their original form and/or genomic locus, or the promoteris not the native promoter for the operably linked polynucleotide.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved. The expression cassette can alsocomprise a selectable marker gene for the selection of transformedcells. Selectable marker genes are utilized for the selection oftransformed cells or tissues.

Isolated polynucleotides are provided that can be used in variousmethods for the detection and/or identification of the soybeanDP-305423-1 event. An “isolated” or “purified” polynucleotide, orbiologically active portion thereof, is substantially or essentiallyfree from components that normally accompany or interact with thepolynucleotide as found in its naturally occurring environment. Thus, anisolated or purified polynucleotide is substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. Optimally, an “isolated”polynucleotide is free of sequences (optimally protein encodingsequences) that naturally flank the polynucleotide (i.e., sequenceslocated at the 5′ and 3′ ends of the polynucleotide) in the genomic DNAof the organism from which the polynucleotide is derived. For example,in various embodiments, the isolated polynucleotide can contain lessthan about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotidesequence that naturally flank the polynucleotide in genomic DNA of thecell from which the polynucleotide is derived.

In specific embodiments, the polynucleotides of the invention comprisethe junction DNA sequence set forth in SEQ ID NO:8, 9, 14, 15, 20, 21,83 or 84. In other embodiments, the polynucleotides of the inventioncomprise the junction DNA sequences set forth in SEQ ID NO:5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82,83, 84, 85, 86, 87 or 88 or variants and fragments thereof. Fragmentsand variants of junction DNA sequences are suitable for discriminativelyidentifying event DP-305423-1. As discussed elsewhere herein, suchsequences find use as primer and/or probes.

Another embodiment is a DNA expression construct comprising the isolatedpolynucleotide of the invention operably linked to at least oneregulatory sequence.

Another embodiment is a recombinant DNA construct comprising: a firstand second expression cassette, wherein said first expression cassettein operable linkage comprises: (a) a soybean KTi3 promoter; (b) agm-fad2-1 fragment; and (c) a soybean KTi3 transcriptional terminator;and said second expression cassette comprising in operable linkage: (i)a soybean SAMS promoter; (ii) a soybean SAMS 5′ untranslated leader andintron; (iii) a soybean gm-hra encoding DNA molecule; and (iv) a soybeanals transcriptional terminator.

Another embodiment is a transgenic soybean plant having stablyintegrated into its genome the polynucleotide or the recombinant DNAconstruct of the invention, and transgenic seed and transgenic progenydrived from said transgenic soybean plant, each also comprising thepolynucleotide or recombinant DNA construct of the invention.

In other embodiments, the polynucleotides of the invention comprisepolynucleotides that can detect a DP-305423-1 event or a DP-305423-1specific region. Such sequences include any polynucleotide set forth inSEQ ID NOS:1-94 or variants and fragments thereof. Fragments andvariants of polynucleotides that detect a DP-305423-1 event or aDP-305423-1 specific region are suitable for discriminativelyidentifying event DP-305423-1. As discussed elsewhere herein, suchsequences find us as primer and/or probes. Further provided are isolatedDNA nucleotide primer sequences comprising or consisting of a sequenceset forth in SEQ ID NO:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 89, 90, 91, 92, 93 or 94, or acomplement thereof.

“Variants” is intended to mean substantially similar sequences. Forpolynucleotides, a variant comprises a polynucleotide having deletions(i.e., truncations) at the 5′ and/or 3′ end; deletion and/or addition ofone or more nucleotides at one or more internal sites in the nativepolynucleotide; and/or substitution of one or more nucleotides at one ormore sites in the native polynucleotide.

As used herein, a “probe” is an isolated polynucleotide to which isattached a conventional detectable label or reporter molecule, e.g., aradioactive isotope, ligand, chemiluminescent agent, enzyme, etc. Such aprobe is complementary to a strand of a target polynucleotide, in thecase of the present invention, to a strand of isolated DNA from soybeanevent DP-305423-1 whether from a soybean plant or from a sample thatincludes DNA from the event. Probes according to the present inventioninclude not only deoxyribonucleic or ribonucleic acids but alsopolyamides and other probe materials that can specifically detect thepresence of the target DNA sequence.

As used herein, “primers” are isolated polynucleotides that are annealedto a complementary target DNA strand by nucleic acid hybridization toform a hybrid between the primer and the target DNA strand, thenextended along the target DNA strand by a polymerase, e.g., a DNApolymerase. Primer pairs of the invention refer to their use foramplification of a target polynucleotide, e.g., by the polymerase chainreaction (PCR) or other conventional nucleic-acid amplification methods.“PCR” or “polymerase chain reaction” is a technique used for theamplification of specific DNA segments (see, U.S. Pat. Nos. 4,683,195and 4,800,159; herein incorporated by reference). Any combination ofprimers disclosed herein can be used such that the pair allows for thedetection a DP-305423-1 event or specific region. Non-limiting examplesof primer pairs include SEQ ID NOS:26 and 27; SEQ ID NOS:29 and 30; SEQID NOS:31 and 32; SEQ ID NOS:33 AND 34; SEQ ID NOS:35 and 36; SEQ IDNOS:37 and 38; SEQ ID NOS:39 and 40; SEQ ID NO:41 and 42; SEQ ID NOS:43and 44; SEQ ID NOS:45 and 46; SEQ ID NOS:47 and 48; SEQ ID NOS:47 and49; SEQ ID NOS:50 and 51; SEQ ID NOS:52 and 53; SEQ ID NOS:54 and 49;SEQ ID NOS:55 and 46; SEQ ID NOS:33 and 56; SEQ ID NOS:57 and 58; SEQ IDNOS:59 and 60; SEQ ID NOS:61 and 36; SEQ ID NOS:35 and 62; SEQ ID NOS:37and 63; SEQ ID NOS:64 and 65; SEQ ID NOS:66 and 67; SEQ ID NOS:68 and69; SEQ ID NOS:70 and 71; SEQ ID NOS:72 and 73; SEQ ID NOS:74 and 75;SEQ ID NOS:76 and 77; SEQ ID NOS:78 and 79; SEQ ID NOS:80 and 81; andSEQ ID NOS:89 and 90.

Probes and primers are of sufficient nucleotide length to bind to thetarget DNA sequence and specifically detect and/or identify apolynucleotide having a DP-305423-1 event. It is recognized that thehybridization conditions or reaction conditions can be determined by theoperator to achieve this result. This length may be of any length thatis of sufficient length to be useful in a detection method of choice.Generally, 8, 11, 14, 16, 18, 20, 22, 24, 26, 28, 30, 40, 50, 75, 100,200, 300, 400, 500, 600, 700 nucleotides or more, or between about11-20, 20-30, 30-40, 40-50, 50-100, 100-200, 200-300, 300-400, 400-500,500-600, 600-700, 700-800, or more nucleotides in length are used. Suchprobes and primers can hybridize specifically to a target sequence underhigh stringency hybridization conditions. Probes and primers accordingto embodiments of the present invention may have complete DNA sequenceidentity of contiguous nucleotides with the target sequence, althoughprobes differing from the target DNA sequence and that retain theability to specifically detect and/or identify a target DNA sequence maybe designed by conventional methods. Accordingly, probes and primers canshare about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or greater sequence identity or complementarity to the targetpolynucleotide (i.e., SEQ ID NO:1-94), or can differ from the targetsequence (i.e., SEQ ID NO:1-94) by 1, 2, 3, 4, 5, 6 or more nucleotides.Probes can be used as primers, but are generally designed to bind to thetarget DNA or RNA and are not used in an amplification process.

Specific primers can be used to amplify an integration fragment toproduce an amplicon that can be used as a “specific probe” or can itselfbe detected for identifying event DP-305423-1 in biological samples.Alternatively, a probe of the invention can be used during the PCRreaction to allow for the detection of the amplification event (i.e., ataqman probe). When the probe is hybridized with the polynucleotides ofa biological sample under conditions which allow for the binding of theprobe to the sample, this binding can be detected and thus allow for anindication of the presence of event DP-305423-1 in the biologicalsample. Such identification of a bound probe has been described in theart. In an embodiment of the invention, the specific probe is a sequencewhich, under optimized conditions, hybridizes specifically to a regionwithin the 5′ or 3′ flanking region of the event and also comprises apart of the foreign DNA contiguous therewith. The specific probe maycomprise a sequence of at least 80%, between 80 and 85%, between 85 and90%, between 90 and 95%, and between 95 and 100% identical (orcomplementary) to a specific region of the DP-305423-1 event.

As used herein, “amplified DNA” or “amplicon” refers to the product ofpolynucleotide amplification of a target polynucleotide that is part ofa nucleic acid template. For example, to determine whether a soybeanplant resulting from a sexual cross contains the DP-305423-1 event, DNAextracted from the soybean plant tissue sample may be subjected to apolynucleotide amplification method using a DNA primer pair thatincludes a first primer derived from flanking sequence adjacent to theinsertion site of inserted heterologous DNA, and a second primer derivedfrom the inserted heterologous DNA to produce an amplicon that isdiagnostic for the presence of the DP-305423-1 event DNA. By“diagnostic” for a DP-305423-1 event the use of any method or assaywhich discriminates between the presence or the absence of a DP-305423-1event in a biological sample is intended. Alternatively, the secondprimer may be derived from the flanking sequence. In still otherembodiments, primer pairs can be derived from flanking sequence on bothsides of the inserted DNA so as to produce an amplicon that includes theentire insert polynucleotide of the expression construct as well as thesequence flanking the transgenic insert. The amplicon is of a length andhas a sequence that is also diagnostic for the event (i.e., has ajunction DNA from a DP-305423-1 event). The amplicon may range in lengthfrom the combined length of the primer pairs plus one nucleotide basepair to any length of amplicon producible by a DNA amplificationprotocol. A member of a primer pair derived from the flanking sequencemay be located a distance from the inserted DNA sequence, this distancecan range from one nucleotide base pair up to the limits of theamplification reaction, or about twenty thousand nucleotide base pairs.The use of the term “amplicon” specifically excludes primer dimers thatmay be formed in the DNA thermal amplification reaction.

Methods for preparing and using probes and primers are described, forexample, in Molecular Cloning: A Laboratory Manual, 2.sup.nd ed, vol.1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. 1989 (hereinafter, “Sambrook et al., 1989”); CurrentProtocols in Molecular Biology, ed. Ausubel et al., Greene Publishingand Wiley-Interscience, New York, 1992 (with periodic updates)(hereinafter, “Ausubel et al., 1992”); and Innis et al., PCR Protocols:A Guide to Methods and Applications, Academic Press: San Diego, 1990.PCR primer pairs can be derived from a known sequence, for example, byusing computer programs intended for that purpose such as the PCR primeranalysis tool in Vector NTI version 6 (Informax Inc., Bethesda Md.);PrimerSelect (DNASTAR Inc., Madison, Wis.); and Primer (Version0.5.COPYRGT., 1991, Whitehead Institute for Biomedical Research,Cambridge, Mass.). Additionally, the sequence can be visually scannedand primers manually identified using guidelines known to one of skillin the art.

It is to be understood that as used herein the term “transgenic”includes any cell, cell line, callus, tissue, plant part, or plant, thegenotype of which has been altered by the presence of a heterologousnucleic acid including those transgenics initially so altered as well asthose created by sexual crosses or asexual propagation from the initialtransgenic. The term “transgenic” as used herein does not encompass thealteration of the genome (chromosomal or extra-chromosomal) byconventional plant breeding methods or by naturally occurring eventssuch as random cross-fertilization, non-recombinant viral infection,non-recombinant bacterial transformation, non-recombinant transposition,or spontaneous mutation.

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere et al. (1987) Meth. Enzymol. 143: 277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327: 70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference). Additional transformation methods aredisclosed below.

Thus, isolated polynucleotides of the invention can be incorporated intorecombinant constructs, typically DNA constructs, which are capable ofintroduction into and replication in a host cell. Such a construct canbe a vector that includes a replication system and sequences that arecapable of transcription and translation of a polypeptide-encodingsequence in a given host cell. A number of vectors suitable for stabletransfection of plant cells or for the establishment of transgenicplants have been described in, e.g., Pouwels et al. (1985; Supp. 1987)Cloning Vectors: A Laboratory Manual, Weissbach and Weissbach (1989)Methods for Plant Molecular Biology (Academic Press, New York); andFlevin et al. (1990) Plant Molecular Biology Manual (Kluwer AcademicPublishers). Typically, plant expression vectors include, for example,one or more cloned plant genes under the transcriptional control of 5′and 3′ regulatory sequences and a dominant selectable marker. Such plantexpression vectors also can contain a promoter regulatory region (e.g.,a regulatory region controlling inducible or constitutive,environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

Various methods and compositions for identifying event DP-305423-1 areprovided. Such methods find use in identifying and/or detecting aDP-305423-1 event in any biological material. Such methods include, forexample, methods to confirm seed purity and methods for screening seedsin a seed lot for a DP-305423-1 event. In one embodiment, a method foridentifying event DP-305423-1 in a biological sample is provided andcomprises contacting the sample with a first and a second primer; and,amplifying a polynucleotide comprising a DP-305423-1 specific region.

A biological sample can comprise any sample in which one desires todetermine if DNA having event DP-305423-1 is present. For example, abiological sample can comprise any plant material or material comprisingor derived from a plant material such as, but not limited to, food orfeed products. As used herein, “plant material” refers to material whichis obtained or derived from a plant or plant part. In specificembodiments, the biological sample comprises a soybean tissue.

Primers and probes based on the flanking DNA and insert sequencesdisclosed herein can be used to confirm (and, if necessary, to correct)the disclosed sequences by conventional methods, e.g., by re-cloning andsequencing such sequences. The polynucleotide probes and primers of thepresent invention specifically detect a target DNA sequence. Anyconventional nucleic acid hybridization or amplification method can beused to identify the presence of DNA from a transgenic event in asample. By “specifically detect” it is intended that the polynucleotidecan be used either as a primer to amplify a DP-305423-1 specific regionor the polynucleotide can be used as a probe that hybridizes understringent conditions to a polynucleotide having a DP-305423-1 event or aDP-305423-1 specific region. The level or degree of hybridization whichallows for the specific detection of a DP-305423-1 event or a specificregion of a DP-305423-1 event is sufficient to distinguish thepolynucleotide with the DP-305423-1 specific region from apolynucleotide lacking this region and thereby allow for discriminatelyidentifying a DP-305423-1 event. By “shares sufficient sequence identityor complentarity to allow for the amplification of a DP-305423-1specific event” is intended the sequence shares at least 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity orcomplementarity to a fragment or across the full length of thepolynucleotide having the DP-305423-1 specific region.

Regarding the amplification of a target polynucleotide (e.g., by PCR)using a particular amplification primer pair, “stringent conditions” areconditions that permit the primer pair to hybridize to the targetpolynucleotide to which a primer having the corresponding wild-typesequence (or its complement) would bind and preferably to produce anidentifiable amplification product (the amplicon) having a DP-305423-1specific region in a DNA thermal amplification reaction. In a PCRapproach, oligonucleotide primers can be designed for use in PCRreactions to amplify a DP-305423-1 specific region. Methods fordesigning PCR primers and PCR cloning are generally known in the art andare disclosed in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, New York); Innis and Gelfand, eds. (1995)PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds.(1999) PCR Methods Manual (Academic Press, New York). Methods ofamplification are further described in U.S. Pat. Nos. 4,683,195,4,683,202 and Chen et al. (1994) PNAS 91:5695-5699. These methods aswell as other methods known in the art of DNA amplification may be usedin the practice of the embodiments of the present invention. It isunderstood that a number of parameters in a specific PCR protocol mayneed to be adjusted to specific laboratory conditions and may beslightly modified and yet allow for the collection of similar results.These adjustments will be apparent to a person skilled in the art.

The amplified polynucleotide (amplicon) can be of any length that allowsfor the detection of the DP-305423-1 event or a DP-305423-1 specificregion. For example, the amplicon can be about 10, 50, 100, 200, 300,500, 700, 100, 2000, 3000, 4000, 5000 nucleotides in length or longer.

In specific embodiments, the specific region of the DP-305423-1 event isdetected.

Any primer can be employed in the methods of the invention that allows aDP-305423-1 specific region to be amplified and/or detected. Forexample, in specific embodiments, the first primer comprises a fragmentof a polynucleotide of SEQ ID NO:5, 6, 7 or 82, wherein the first or thesecond primer shares sufficient sequence identity or complementarity tothe polynucleotide to amplify the DP-305423-1 specific region. Theprimer pair can comprise a first primer that comprises a fragment of a5′ genomic region of SEQ ID NO:5, 6, 7 or 82, and a second primer thatcomprises a fragment of a 3′ genomic region of SEQ ID NO:5, 6, 7 or 82,or an insert region of SEQ ID NO:5, 6, 7 or 82, or alternatively, theprimer pair can comprise a first primer that comprises a fragment of a3′ genomic region of SEQ ID NO:5, 6, 7 or 82, and a second primer thatcomprises a fragment of a 5′ genomic region of SEQ ID NO:5, 6, 7 or 82,or an insert region of SEQ ID NO:5, 6, 7 or 82. In still furtherembodiments, the first and the second primer can comprise any one or anycombination of the sequences set forth in SEQ ID NO:26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 89, 90, 91,92, 93 or 94. The primers can be of any length sufficient to amplify aDP-305423-1 region including, for example, at least 6, 7, 8, 9, 10, 15,20, 15, or 30 or about 7-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40,40-45 nucleotides or longer.

As discussed elsewhere herein, any method to PCR amplify the DP-305423-1event or specific region can be employed, including for example, realtime PCR. See, for example, Livak et al. (1995a) Oligonucleotides withfluorescent dyes at opposite ends provide a quenched probe system fordetecting PCR product and nucleic acid hybridization. PCR methods andApplications. 4:357-362; U.S. Pat. No. 5,538,848; U.S. Pat. No.5,723,591; Applied Biosystems User Bulletin No. 2, “RelativeQuantitation of Gene Expression,” U.S. Pat. No. 4,303,859; and, AppliedBiosystems User Bulletin No. 5, “Multiplex PCR with Taqman VIC probes,”U.S. Pat. No. 4,306,236; each of which is herein incorporated byreference.

Thus, in specific embodiments, a method of detecting the presence ofsoybean event DP-305423-1 or progeny thereof in a biological sample isprovided. The method comprises (a) extracting a DNA sample from thebiological sample; (b) providing a pair of DNA primer molecules,including, but not limited to, i) the sequences comprising SEQ ID NO:26and SEQ ID NO:27; ii) the sequences comprising SEQ ID NO:29 and SEQ IDNO:30; iii) the sequences comprising SEQ ID NO:31 and SEQ ID NO:32; iv)the sequences comprising SEQ ID NO:33 and SEQ ID NO:34; v) the sequencescomprising SEQ ID NO:35 and SEQ ID NO:36; vi) the sequences comprisingSEQ ID NO:37 and SEQ ID NO:38; vii) the sequences comprising SEQ IDNO:39 and SEQ ID NO:40; viii) the sequences comprising SEQ ID NO:41 andSEQ ID NO:42; ix) the sequences comprising SEQ ID NO:43 and SEQ IDNO:44; x) the sequences comprising SEQ ID NO:45 and SEQ ID NO:46; xi)the sequences comprising SEQ ID NO:47 and SEQ ID NO:48; xii) thesequences comprising SEQ ID NO:47 and SEQ ID NO:49; xiii) the sequencescomprising SEQ ID NO:50 and SEQ ID NO:51; xiv) the sequences comprisingSEQ ID NO:52 and SEQ ID NO:53; xv) the sequences comprising SEQ ID NO:54and SEQ ID NO:49; xvi) the sequences comprising SEQ ID NO:55 and SEQ IDNO:46; xvii) the sequences comprising SEQ ID NO:33 and SEQ ID NO:56;xviii) the sequences comprising SEQ ID NO:57 and SEQ ID NO:58; xix) thesequences comprising SEQ ID NO:59 and SEQ ID NO:60; xx) the sequencescomprising SEQ ID NO:61 and SEQ ID NO:36; xxi) the sequences comprisingSEQ ID NO:35 and SEQ ID NO:62; xxii) the sequences comprising SEQ IDNO:37 and SEQ ID NO:63; xxiii) the sequences comprising SEQ ID NO:64 andSEQ ID NO:65; xxiv) the sequences comprising SEQ ID NO:66 and SEQ IDNO:67; xxv) the sequences comprising SEQ ID NO:68 and SEQ ID NO:69;xxvi) the sequences comprising SEQ ID NO:70 and SEQ ID NO:71; xxvii) thesequences comprising SEQ ID NO:72 and SEQ ID NO:73; xxviii) thesequences comprising SEQ ID NO:74 and SEQ ID NO:75; xxix) the sequencescomprising SEQ ID NO:76 and SEQ ID NO:77; xxx) the sequences comprisingSEQ ID NO:78 and SEQ ID NO:79; xxxi) the sequences comprising SEQ IDNO:80 and SEQ ID NO:81; and xxxii) the sequences comprising SEQ ID NO:89and SEQ ID NO:90 (c) providing DNA amplification reaction conditions;(d) performing the DNA amplification reaction, thereby producing a DNAamplicon molecule; and (e) detecting the DNA amplicon molecule, whereinthe detection of said DNA amplicon molecule in the DNA amplificationreaction indicates the presence of soybean event DP-305423-1. In orderfor a nucleic acid molecule to serve as a primer or probe it need onlybe sufficiently complementary in sequence to be able to form a stabledouble-stranded structure under the particular solvent and saltconcentrations employed.

In hybridization techniques, all or part of a polynucleotide thatselectively hybridizes to a target polynucleotide having a DP-305423-1specific event is employed. By “stringent conditions” or “stringenthybridization conditions” when referring to a polynucleotide probeconditions under which a probe will hybridize to its target sequence toa detectably greater degree than to other sequences (e.g., at least2-fold over background) are intended. Regarding the amplification of atarget polynucleotide (e.g., by PCR) using a particular amplificationprimer pair, “stringent conditions” are conditions that permit theprimer pair to hybridize to the target polynucleotide to which a primerhaving the corresponding wild-type. Stringent conditions aresequence-dependent and will be different in different circumstances. Bycontrolling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of identity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length or lessthan 500 nucleotides in length.

As used herein, a substantially identical or complementary sequence is apolynucleotide that will specifically hybridize to the complement of thenucleic acid molecule to which it is being compared under highstringency conditions. Appropriate stringency conditions which promoteDNA hybridization, for example, 6× sodium chloride/sodium citrate (SSC)at about 45° C., followed by a wash of 2×SSC at 50° C., are known tothose skilled in the art or can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.Typically, stringent conditions for hybridization and detection will bethose in which the salt concentration is less than about 1.5 M Na ion,typically about 0.01 to 1.0 M Na ion concentration (or other salts) atpH 7.0 to 8.3 and the temperature is at least about 30° C. for shortprobes (e.g., 10 to 50 nucleotides) and at least about 60° C. for longprobes (e.g., greater than 50 nucleotides). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide. Exemplary low stringency conditions include hybridizationwith a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodiumdodecyl sulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 MNaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderatestringency conditions include hybridization in 40 to 45% formamide, 1.0M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C.Exemplary high stringency conditions include hybridization in 50%formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to65° C. Optionally, wash buffers may comprise about 0.1% to about 1% SDS.Duration of hybridization is generally less than about 24 hours, usuallyabout 4 to about 12 hours. The duration of the wash time will be atleast a length of time sufficient to reach equilibrium.

In hybridization reactions, specificity is typically the function ofpost-hybridization washes, the critical factors being the ionic strengthand temperature of the final wash solution. For DNA-DNA hybrids, theT_(m) can be approximated from the equation of Meinkoth and Wahl (1984)Anal. Biochem. 138:267-284: T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61(% form)−500/L; where M is the molarity of monovalent cations, % GC isthe percentage of guanosine and cytosine nucleotides in the DNA, % formis the percentage of formamide in the hybridization solution, and L isthe length of the hybrid in base pairs. The T_(m) is the temperature(under defined ionic strength and pH) at which 50% of a complementarytarget sequence hybridizes to a perfectly matched probe. T_(m) isreduced by about 1° C. for each 1% of mismatching; thus, T_(m),hybridization, and/or wash conditions can be adjusted to hybridize tosequences of the desired identity. For example, if sequences with >90%identity are sought, the T_(m) can be decreased 10° C. Generally,stringent conditions are selected to be about 5° C. lower than thethermal melting point (T_(m)) for the specific sequence and itscomplement at a defined ionic strength and pH. However, severelystringent conditions can utilize a hybridization and/or wash at 1, 2, 3,or 4° C. lower than the thermal melting point (T_(m)); moderatelystringent conditions can utilize a hybridization and/or wash at 6, 7, 8,9, or 10° C. lower than the thermal melting point (T_(m)); lowstringency conditions can utilize a hybridization and/or wash at 11, 12,13, 14, 15, or 20° C. lower than the thermal melting point (T_(m)).Using the equation, hybridization and wash compositions, and desiredT_(m), those of ordinary skill will understand that variations in thestringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a T_(m) ofless than 45° C. (aqueous solution) or 32° C. (formamide solution), itis optimal to increase the SSC concentration so that a highertemperature can be used. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds.(1995) Current Protocols in Molecular Biology, Chapter 2 (GreenePublishing and Wiley-Interscience, New York). See Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.) and Haymes et al. (1985) In: NucleicAcid Hybridization, a Practical Approach, IRL Press, Washington, D.C.

A polynucleotide is said to be the “complement” of anotherpolynucleotide if they exhibit complementarity. As used herein,molecules are said to exhibit “complete complementarity” when everynucleotide of one of the polynucleotide molecules is complementary to anucleotide of the other. Two molecules are said to be “minimallycomplementary” if they can hybridize to one another with sufficientstability to permit them to remain annealed to one another under atleast conventional “low-stringency” conditions. Similarly, the moleculesare said to be “complementary” if they can hybridize to one another withsufficient stability to permit them to remain annealed to one anotherunder conventional “high-stringency” conditions.

Further provided are methods of detecting the presence of DNAcorresponding to the DP-305423-1 event in a sample. In one embodiment,the method comprises (a) contacting the biological sample with apolynucleotide probe that hybridizes under stringent hybridizationconditions with DNA from soybean event DP-305423-1 and specificallydetects the DP-305423-1 event; (b) subjecting the sample and probe tostringent hybridization conditions; and (c) detecting hybridization ofthe probe to the DNA, wherein detection of hybridization indicates thepresence of the DP-305423-1 event.

Various method can be used to detect the DP-305423-1 specific region oramplicon thereof, including, but not limited to, Genetic Bit Analysis(Nikiforov et al. (1994) Nucleic Acid Res. 22: 4167-4175) where a DNAoligonucleotide is designed which overlaps both the adjacent flankingDNA sequence and the inserted DNA sequence. The oligonucleotide isimmobilized in wells of a microwell plate. Following PCR of the regionof interest (using one primer in the inserted sequence and one in theadjacent flanking sequence) a single-stranded PCR product can behybridized to the immobilized oligonucleotide and serve as a templatefor a single base extension reaction using a DNA polymerase and labeledddNTPs specific for the expected next base. Readout may be fluorescentor ELISA-based. A signal indicates presence of the insert/flankingsequence due to successful amplification, hybridization, and single baseextension.

Another detection method is the Pyrosequencing technique as described byWinge ((2000) Innov. Pharma. Tech. 00: 18-24). In this method, anoligonucleotide is designed that overlaps the adjacent DNA and insertDNA junction. The oligonucleotide is hybridized to a single-stranded PCRproduct from the region of interest (one primer in the inserted sequenceand one in the flanking sequence) and incubated in the presence of a DNApolymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5′phosphosulfate and luciferin. dNTPs are added individually and theincorporation results in a light signal which is measured. A lightsignal indicates the presence of the transgene insert/flanking sequencedue to successful amplification, hybridization, and single or multi-baseextension.

Fluorescence Polarization as described by Chen et al. ((1999) GenomeRes. 9: 492-498, 1999) is also a method that can be used to detect anamplicon of the invention. Using this method, an oligonucleotide isdesigned which overlaps the flanking and inserted DNA junction. Theoligonucleotide is hybridized to a single-stranded PCR product from theregion of interest (one primer in the inserted DNA and one in theflanking DNA sequence) and incubated in the presence of a DNA polymeraseand a fluorescent-labeled ddNTP. Single base extension results inincorporation of the ddNTP. Incorporation can be measured as a change inpolarization using a fluorometer. A change in polarization indicates thepresence of the transgene insert/flanking sequence due to successfulamplification, hybridization, and single base extension.

Taqman® (PE Applied Biosystems, Foster City, Calif.) is described as amethod of detecting and quantifying the presence of a DNA sequence andis fully understood in the instructions provided by the manufacturer.Briefly, a FRET oligonucleotide probe is designed which overlaps theflanking and insert DNA junction. The FRET probe and PCR primers (oneprimer in the insert DNA sequence and one in the flanking genomicsequence) are cycled in the presence of a thermostable polymerase anddNTPs. Hybridization of the FRET probe results in cleavage and releaseof the fluorescent moiety away from the quenching moiety on the FRETprobe. A fluorescent signal indicates the presence of theflanking/transgene insert sequence due to successful amplification andhybridization.

Molecular Beacons have been described for use in sequence detection asdescribed in Tyangi et al. ((1996) Nature Biotech. 14: 303-308).Briefly, a FRET oligonucleotide probe is designed that overlaps theflanking and insert DNA junction. The unique structure of the FRET proberesults in it containing secondary structure that keeps the fluorescentand quenching moieties in close proximity. The FRET probe and PCRprimers (one primer in the insert DNA sequence and one in the flankingsequence) are cycled in the presence of a thermostable polymerase anddNTPs. Following successful PCR amplification, hybridization of the FRETprobe to the target sequence results in the removal of the probesecondary structure and spatial separation of the fluorescent andquenching moieties. A fluorescent signal results. A fluorescent signalindicates the presence of the flanking/transgene insert sequence due tosuccessful amplification and hybridization.

A hybridization reaction using a probe specific to a sequence foundwithin the amplicon is yet another method used to detect the ampliconproduced by a PCR reaction.

As used herein, “kit” refers to a set of reagents for the purpose ofperforming the method embodiments of the invention, more particularly,the identification and/or the detection of the DP-305423-1 event inbiological samples. The kit of the invention can be used, and itscomponents can be specifically adjusted, for purposes of quality control(e.g. purity of seed lots), detection of event DP-305423-1 in plantmaterial, or material comprising or derived from plant material, such asbut not limited to food or feed products.

In specific embodiments, a kit for identifying event DP-305423-1 in abiological sample is provided. The kit comprises a first and a secondprimer, wherein the first and second primer amplify a polynucleotidecomprising a DP-305423-1 specific region. In further embodiments, thekit also comprises a polynucleotide for the detection of the DP-305423-1specific region. The kit can comprise, for example, a first primercomprising a fragment of a polynucleotide of SEQ ID NO:5, 6, 7 or 82,wherein the first or the second primer shares sufficient sequencehomology or complementarity to the polynucleotide to amplify saidDP-305423-1 specific region. For example, in specific embodiments, thefirst primer comprises a fragment of a polynucleotide of SEQ ID NO:5, 6,7 or 82, wherein the first or the second primer shares sufficientsequence homology or complementarity to the polynucleotide to amplifysaid DP-305423-1 specific region. The primer pair can comprises a firstprimer that comprises a fragment of a 5′ genomic region of SEQ ID NO:5,6, 7 or 82, and a second primer that comprises a fragment of a 3′genomic region of SEQ ID NO:5, 6, 7 or 82, or an insert region of SEQ IDNO:5, 6, 7 or 82, or alternatively, the primer pair can comprise a firstprimer that comprises a fragment of a 3′ genomic region of SEQ ID NO:5,6, 7 or 82, and a second primer that comprises a fragment of a 5′genomic region of SEQ ID NO:5, 6, 7 or 82, or an insert region of SEQ IDNO:5, 6, 7 or 82. In still further embodiments, the first and the secondprimer can comprise any one or any combination of the sequences setforth in SEQ ID NO:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 89, 90, 91, 92, 93 or 94. The primers can beof any length sufficient to amplify the DP-305423-1 region including,for example, at least 6, 7, 8, 9, 10, 15, 20, 15, or 30 or about 7-10,10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45 nucleotides or longer.

Further provided are DNA detection kits comprising at least onepolynucleotide that can specifically detect a DP-305423-1 specificregion, wherein said polynucleotide comprises at least one DNA moleculeof a sufficient length of contiguous nucleotides homologous orcomplementary to SEQ ID NO:5, 6, 7 or 82. In specific embodiments, theDNA detection kit comprises a polynucleotide having SEQ ID NO:8, 9, 14,15, 20, 21, 83 or 84, or comprises a sequence which hybridizes with atleast one sequence selected from the group consisting of: a) thesequences of a 5′ genomic region of SEQ ID NO:5, 6, 7 or 82, and thesequences of an insert region of SEQ ID NO:5, 6, 7 or 82; and, b) thesequences of a 3′ genomic region of SEQ ID NO:5, 6, 7 or 82, and thesequences of an insert region of SEQ ID NO:5, 6, 7 or 82.

Any of the polynucleotides and fragments and variants thereof employedin the methods and compositions of the invention can share sequenceidentity to a region of the transgene insert of the DP-305423-1 event, ajunction sequence of the DP-305423-1 event or a flanking sequence of theDP-305423-1 event. Methods to determine the relationship of varioussequences are known. As used herein, “reference sequence” is a definedsequence used as a basis for sequence comparison. A reference sequencemay be a subset or the entirety of a specified sequence; for example, asa segment of a full-length cDNA or gene sequence, or the complete cDNAor gene sequence. As used herein, “comparison window” makes reference toa contiguous and specified segment of a polynucleotide sequence, whereinthe polynucleotide sequence in the comparison window may compriseadditions or deletions (i.e., gaps) compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two polynucleotides. Generally, the comparison window is at least20 contiguous nucleotides in length, and optionally can be 30, 40, 50,100, or longer. Those of skill in the art understand that to avoid ahigh similarity to a reference sequence due to inclusion of gaps in thepolynucleotide sequence a gap penalty is typically introduced and issubtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent sequence identity between anytwo sequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-local alignment method of Pearson and Lipman (1988) Proc.Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the GCG Wisconsin Genetics Software Package, Version 10(available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153;Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992)CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.The ALIGN program is based on the algorithm of Myers and Miller (1988)supra. A PAM120 weight residue table, a gap length penalty of 12, and agap penalty of 4 can be used with the ALIGN program when comparing aminoacid sequences. The BLAST programs of Altschul et al (1990) J. Mol.Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990)supra. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a nucleotide sequence encoding a protein of the invention.BLAST protein searches can be performed with the BLASTX program,score=50, wordlength=3, to obtain amino acid sequences homologous to aprotein or polypeptide of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389.Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform aniterated search that detects distant relationships between molecules.See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST,PSI-BLAST, the default parameters of the respective programs (e.g.,BLASTN for nucleotide sequences, BLASTX for proteins) can be used. Seewww.ncbi.nlm.nih.gov. Alignment may also be performed manually byinspection.

Sequence alignments and percent identity calculations may be determinedusing a variety of comparison methods designed to detect homologoussequences including, but not limited to, the Megalign® program of theLASERGENE® bioinformatics computing suite (DNASTAR® Inc., Madison,Wis.). For example, multiple alignment of the sequences provided hereincan be performed using the Clustal V method of alignment (Higgins andSharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments and calculation of percent identity of protein sequencesusing the Clustal V method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 andDIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAPPENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of thesequences, using the Clustal V program, it is possible to obtain“percent identity” and “divergence” values by viewing the “sequencedistances” table on the same program.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix; or any equivalent program thereof. By“equivalent program” any sequence comparison program that, for any twosequences in question, generates an alignment having identicalnucleotide or amino acid residue matches and an identical percentsequence identity when compared to the corresponding alignment generatedby GAP Version 10 is intended.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol.48:443-453, to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the GCG Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 200. Thus, for example, the gapcreation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the Quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the GCG Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

As used herein, “sequence identity” or “identity” in the context of twopolynucleotides or polypeptide sequences makes reference to the residuesin the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

The present invention provides methods for controlling weeds in an areaof cultivation, preventing the development or the appearance ofherbicide resistant weeds in an area of cultivation, producing a crop,and increasing crop safety. The term “controlling,” and derivationsthereof, for example, as in “controlling weeds” refers to one or more ofinhibiting the growth, germination, reproduction, and/or proliferationof; and/or killing, removing, destroying, or otherwise diminishing theoccurrence and/or activity of a weed.

As used herein, an “area of cultivation” comprises any region in whichone desires to grow a plant. Such areas of cultivations include, but arenot limited to, a field in which a plant is cultivated (such as a cropfield, a sod field, a tree field, a managed forest, a field forculturing fruits and vegetables, etc), a greenhouse, a growth chamber,etc.

The methods of the invention comprise planting the area of cultivationwith the soybean DP-305423-1 seeds or plants, and in specificembodiments, applying to the crop, seed, weed or area of cultivationthereof an effective amount of a herbicide of interest. It is recognizedthat the herbicide can be applied before or after the crop is planted inthe area of cultivation. Such herbicide applications can include anapplication of an inhibitor of ALS. In specific embodiments, aninhibitor of ALS is applied to the soybean DP-305423-1 event, whereinthe effective concentration of the ALS inhibitor would significantlydamage an appropriate control plant. In one non-limiting embodiment, theherbicide comprises at least one of a sulfonylaminocarbonyltriazolinone;a triazolopyrimidine; a pyrimidinyl(thio)benzoate; an imidazolinone; atriazine; and/or a phosphinic acid.

In another non-limiting embodiment, the herbicide comprises imazapyr,chlorimuron-ethyl, quizalofop, or fomesafen, wherein an effective amountis tolerated by the crop and controls weeds. As disclosed elsewhereherein, any effective amount of these herbicides can be applied. Inspecific embodiments, an effective amount of imazapyr comprising about7.5 to about 27.5 g ai/hectare; an effective amount of chlorimuron-ethylcomprising about 7.5 to about 27.5 g ai/hectare; an effective amount ofquizalofop comprising about 50 to about 70 g ai/hectare; and, aneffective amount of fomesafen comprising about 240 to about 260 gai/hectare.

In other embodiments, a combination of at least two herbicides areapplied. More details regarding the various herbicide combinations thatcan be employed in the methods of the invention are discussed elsewhereherein.

A “control” or “control plant” or “control plant cell” provides areference point for measuring changes in phenotype of the subject plantor plant cell, and may be any suitable plant or plant cell. A controlplant or plant cell may comprise, for example: (a) a wild-type plant orcell, i.e., of the same genotype as the starting material for thegenetic alteration which resulted in the subject plant or cell; (b) aplant or plant cell of the same genotype as the starting material butwhich has been transformed with a null construct (i.e., with a constructwhich has no known effect on the trait of interest, such as a constructcomprising a marker gene); (c) a plant or plant cell which is anon-transformed segregant among progeny of a subject plant or plantcell; (d) a plant or plant cell which is genetically identical to thesubject plant or plant cell but which is not exposed to the sametreatment (e.g., herbicide treatment) as the subject plant or plantcell; (e) the subject plant or plant cell itself, under conditions inwhich the gene of interest is not expressed; or (f) the subject plant orplant cell itself, under conditions in which it has not been exposed toa particular treatment such as, for example, a herbicide or combinationof herbicides and/or other chemicals. In some instances, an appropriatecontrol plant or control plant cell may have a different genotype fromthe subject plant or plant cell but may share the herbicide-sensitivecharacteristics of the starting material for the genetic alteration(s)which resulted in the subject plant or cell (see, e.g., Green (1998)Weed Technology 12: 474-477; Green and Ulrich (1993) Weed Science 41:508-516. In some instances, an appropriate control soybean plant is a“Jack” soybean plant (Illinois Foundation Seed, Champaign, Ill.). Inother embodiments, the null segregant can be used as a control, as theyare genetically identical to DP-305423-1 with the exception of thetransgenic insert DNA.

Any herbicide can be applied to the DP-305423-1 soybean crop, crop part,or the area of cultivation containing the crop plant. Classifications ofherbicides (i.e., the grouping of herbicides into classes andsubclasses) is well-known in the art and includes classifications byHRAC (Herbicide Resistance Action Committee) and WSSA (the Weed ScienceSociety of America) (see also, Retzinger and Mallory-Smith (1997) WeedTechnology 11: 384-393). An abbreviated version of the HRACclassification (with notes regarding the corresponding WSSA group) isset forth below in Table 2.

Herbicides can be classified by their mode of action and/or site ofaction and can also be classified by the time at which they are applied(e.g., preemergent or postemergent), by the method of application (e.g.,foliar application or soil application), or by how they are taken up byor affect the plant. For example, thifensulfuron-methyl andtribenuron-methyl are applied to the foliage of a crop and are generallymetabolized there, while rimsulfuron and chlorimuron-ethyl are generallytaken up through both the roots and foliage of a plant. “Mode of action”generally refers to the metabolic or physiological process within theplant that the herbicide inhibits or otherwise impairs, whereas “site ofaction” generally refers to the physical location or biochemical sitewithin the plant where the herbicide acts or directly interacts.Herbicides can be classified in various ways, including by mode ofaction and/or site of action (see, e.g., Table 2).

Often, a herbicide-tolerance gene that confers tolerance to a particularherbicide or other chemical on a plant expressing it will also confertolerance to other herbicides or chemicals in the same class orsubclass, for example, a class or subclass set forth in Table 2. Thus,in some embodiments of the invention, a transgenic plant of theinvention is tolerant to more than one herbicide or chemical in the sameclass or subclass, such as, for example, an inhibitor of PPO, asulfonylurea, or a synthetic auxin.

The invention provides a transgenic soybean plant which can be selectedfor use in crop production based on the prevalence of herbicide-tolerantweed species in the area where the transgenic crop is to be grown.Methods are known in the art for assessing the herbicide tolerance ofvarious weed species. Weed management techniques are also known in theart, such as for example, crop rotation using a crop that is tolerant toa herbicide to which the local weed species are not tolerant. A numberof entities monitor and publicly report the incidence andcharacteristics of herbicide-tolerant weeds, including the HerbicideResistance Action Committee (HRAC), the Weed Science Society of America,and various state agencies (see, e.g., see, for example, herbicidetolerance scores for various broadleaf weeds from the 2004 IllinoisAgricultural Pest Management Handbook), and one of skill in the artwould be able to use this information to determine which crop andherbicide combinations should be used in a particular location.

These entities also publish advice and guidelines for preventing thedevelopment and/or appearance of and controlling the spread of herbicidetolerant weeds (see, e.g., Owen and Hartzler (2004), 2005 HerbicideManual for Agricultural Professionals, Pub. WC 92 Revised (Iowa StateUniversity Extension, Iowa State University of Science and Technology,Ames, Iowa); Weed Control for Corn, Soybeans, and Sorghum, Chapter 2 of“2004 Illinois Agricultural Pest Management Handbook” (University ofIllinois Extension, University of Illinois at Urbana-Champaign, Ill.);Weed Control Guide for Field Crops, MSU Extension Bulletin E434(Michigan State University, East Lansing, Mich.)).

TABLE 2 Abbreviated version of HRAC Herbicide Classification I. ALSInhibitors (WSSA Group 2) A. Sulfonylureas 1. Azimsulfuron 2.Chlorimuron-ethyl 3. Metsulfuron-methyl 4. Nicosulfuron 5. Rimsulfuron6. Sulfometuron-methyl 7. Thifensulfuron-methyl 8. Tribenuron-methyl 9.Amidosulfuron 10. Bensulfuron-methyl 11. Chlorsulfuron 12. Cinosulfuron13. Cyclosulfamuron 14. Ethametsulfuron-methyl 15. Ethoxysulfuron 16.Flazasulfuron 17. Flupyrsulfuron-methyl 18. Foramsulfuron 19.Imazosulfuron 20. Iodosulfuron-methyl 21. Mesosulfuron-methyl 22.Oxasulfuron 23. Primisulfuron-methyl 24. Prosulfuron 25.Pyrazosulfuron-ethyl 26. Sulfosulfuron 27. Triasulfuron 28.Trifloxysulfuron 29. Triflusulfuron-methyl 30. Tritosulfuron 31.Halosulfuron-methyl 32. Flucetosulfuron B.Sulfonylaminocarbonyltriazolinones 1. Flucarbazone 2. Procarbazone C.Triazolopyrimidines 1. Cloransulam-methyl 2. Flumetsulam 3. Diclosulam4. Florasulam 5. Metosulam 6. Penoxsulam 7. Pyroxsulam D.Pyrimidinyloxy(thio)benzoates 1. Bispyribac 2. Pyriftalid 3.Pyribenzoxim 4. Pyrithiobac 5. Pyriminobac-methyl E. Imidazolinones 1.Imazapyr 2. Imazethapyr 3. Imazaquin 4. Imazapic 5.Imazamethabenz-methyl 6. Imazamox II. Other Herbicides--ActiveIngredients/Additional Modes of Action A. Inhibitors of Acetyl CoAcarboxylase (ACCase) (WSSA Group 1) 1. Aryloxyphenoxypropionates(‘FOPs’) a. Quizalofop-P-ethyl b. Diclofop-methyl c.Clodinafop-propargyl d. Fenoxaprop-P-ethyl e. Fluazifop-P-butyl f.Propaquizafop g. Haloxyfop-P-methyl h. Cyhalofop-butyl i.Quizalofop-P-ethyl 2. Cyclohexanediones (‘DIMs’) a. Alloxydim b.Butroxydim c. Clethodim d. Cycloxydim e. Sethoxydim f. Tepraloxydim g.Tralkoxydim B. Inhibitors of Photosystem II-HRAC Group C1/WSSA Group5 1. Triazines a. Ametryne b. Atrazine c. Cyanazine d. Desmetryne e.Dimethametryne f. Prometon g. Prometryne h. Propazine i. Simazine j.Simetryne k. Terbumeton l. Terbuthylazine m. Terbutryne n. Trietazine 2.Triazinones a. Hexazinone b. Metribuzin c. Metamitron 3. Triazolinone a.Amicarbazone 4. Uracils a. Bromacil b. Lenacil c. Terbacil 5.Pyridazinones a. Pyrazon 6. Phenyl carbamates a. Desmedipham b.Phenmedipham C. Inhibitors of Photosystem II--HRAC Group C2/WSSA Group7 1. Ureas a. Fluometuron b. Linuron c. Chlorobromuron d. Chlorotolurone. Chloroxuron f. Dimefuron g. Diuron h. Ethidimuron i. Fenuron j.Isoproturon k. Isouron l. Methabenzthiazuron m. Metobromuron n.Metoxuron o. Monolinuron p. Neburon q. Siduron r. Tebuthiuron 2. Amidesa. Propanil b. Pentanochlor D. Inhibitors of Photosystem II--HRAC GroupC3/WSSA Group 6 1. Nitriles a. Bromofenoxim b. Bromoxynil c. Ioxynil 2.Benzothiadiazinone (Bentazon) a. Bentazon 3. Phenylpyridazines a.Pyridate b. Pyridafol E. Photosystem-I-electron diversion(Bipyridyliums) (WSSA Group 22) 1. Diquat 2. Paraquat F. Inhibitors ofPPO (protoporphyrinogen oxidase) (WSSA Group 14) 1. Diphenylethers a.Acifluorfen-Na b. Bifenox c. Chlomethoxyfen d. Fluoroglycofen-ethyl e.Fomesafen f. Halosafen g. Lactofen h. Oxyfluorfen 2. Phenylpyrazoles a.Fluazolate b. Pyraflufen-ethyl 3. N-phenylphthalimides a. Cinidon-ethylb. Flumioxazin c. Flumiclorac-pentyl 4. Thiadiazoles a.Fluthiacet-methyl b. Thidiazimin 5. Oxadiazoles a. Oxadiazon b.Oxadiargyl 6. Triazolinones a. Carfentrazone-ethyl b. Sulfentrazone 7.Oxazolidinediones a. Pentoxazone 8. Pyrimidindiones a. Benzfendizone b.Butafenicil 9. Others a. Pyrazogyl b. Profluazol G. Bleaching:Inhibition of carotenoid biosynthesis at the phytoene desaturase step(PDS) (WSSA Group 12) 1. Pyridazinones a. Norflurazon 2.Pyridinecarboxamides a. Diflufenican b. Picolinafen 3. Others a.Beflubutamid b. Fluridone c. Flurochloridone d. Flurtamone H. Bleaching:Inhibition of 4-hydroxyphenyl-pyruvate-dioxygenase (4-HPPD) (WSSA Group28) 1. Triketones a. Mesotrione b. Sulcotrione 2. Isoxazoles a.Isoxachlortole b. Isoxaflutole 3. Pyrazoles a. Benzofenap b. Pyrazoxyfenc. Pyrazolynate 4. Others a. Benzobicyclon I. Bleaching: Inhibition ofcarotenoid biosynthesis (unknown target) (WSSA Group 11 and 13) 1.Triazoles (WSSA Group 11) a. Amitrole 2. Isoxazolidinones (WSSA Group13) a. Clomazone 3. Ureas a. Fluometuron 4. Diphenylether a. AclonifenJ. Inhibition of EPSP Synthase 1. Glycines (WSSA Group 9) a. Glyphosateb. Sulfosate K. Inhibition of glutamine synthetase 1. Phosphinic Acidsa. Glufosinate-ammonium b. Bialaphos L. Inhibition of DHP(dihydropteroate) synthase (WSSA Group 18) 1. Carbamates a. Asulam M.Microtubule Assembly Inhibition (WSSA Group 3) 1. Dinitroanilines a.Benfluralin b. Butralin c. Dinitramine d. Ethalfluralin e. Oryzalin f.Pendimethalin g. Trifluralin 2. Phosphoroamidates a. Amiprophos-methylb. Butamiphos 3. Pyridines a. Dithiopyr b. Thiazopyr 4. Benzamides a.Pronamide b. Tebutam 5. Benzenedicarboxylic acids a. Chlorthal-dimethylN. Inhibition of mitosis/microtubule organization WSSA Group 23) 1.Carbamates a. Chlorpropham b. Propham c. Carbetamide O. Inhibition ofcell division (Inhibition of very long chain fatty acids as proposedmechanism; WSSA Group 15) 1. Chloroacetamides a. Acetochlor b. Alachlorc. Butachlor d. Dimethachlor e. Dimethanamid f. Metazachlor g.Metolachlor h. Pethoxamid i. Pretilachlor j. Propachlor k. Propisochlorl. Thenylchlor 2. Acetamides a. Diphenamid b. Napropamide c.Naproanilide 3. Oxyacetamides a. Flufenacet b. Mefenacet 4.Tetrazolinones a. Fentrazamide 5. Others a. Anilofos b. Cafenstrole c.Indanofan d. Piperophos P. Inhibition of cell wall (cellulose)synthesis 1. Nitriles (WSSA Group 20) a. Dichlobenil b. Chlorthiamid 2.Benzamides (isoxaben (WSSA Group 21)) a. Isoxaben 3.Triazolocarboxamides (flupoxam) a. Flupoxam Q. Uncoupling (membranedisruption): (WSSA Group 24) 1. Dinitrophenols a. DNOC b. Dinoseb c.Dinoterb R. Inhibition of Lipid Synthesis by other than ACCinhibition 1. Thiocarbamates (WSSA Group 8) a. Butylate b. Cycloate c.Dimepiperate d. EPTC e. Esprocarb f. Molinate g. Orbencarb h. Pebulatei. Prosulfocarb j. Benthiocarb k. Tiocarbazil l. Triallate m. Vernolate2. Phosphorodithioates a. Bensulide 3. Benzofurans a. Benfuresate b.Ethofumesate 4. Halogenated alkanoic acids (WSSA Group 26) a. TCA b.Dalapon c. Flupropanate S. Synthetic auxins (IAA-like) (WSSA Group 4) 1.Phenoxycarboxylic acids a. Clomeprop b. 2,4-D c. Mecoprop 2. Benzoicacids a. Dicamba b. Chloramben c. TBA 3. Pyridine carboxylic acids a.Clopyralid b. Fluroxypyr c. Picloram d. Tricyclopyr 4. Quinolinecarboxylic acids a. Quinclorac b. Quinmerac 5. Others (benazolin-ethyl)a. Benazolin-ethyl T. Inhibition of Auxin Transport 1. Phthalamates;semicarbazones (WSSA Group 19) a. Naptalam b. Diflufenzopyr-Na U. OtherMechanism of Action 1. Arylaminopropionic acids a.Flamprop-M-methyl/-isopropyl 2. Pyrazolium a. Difenzoquat 3.Organoarsenicals a. DSMA b. MSMA 4. Others a. Bromobutide b. Cinmethylinc. Cumyluron d. Dazomet e. Daimuron-methyl f. Dimuron g. Etobenzanid h.Fosamine i. Metam j. Oxaziclomefone k. Oleic acid l. Pelargonic acid m.Pyributicarb

In one embodiment, one ALS inhibitor or at least two ALS inhibitors areapplied to the DP-305423-1 soybean crop or area of cultivation. The ALSinhibitor can be applied at any effective rate that selectively controlsweeds and does not significantly damage the crop. In specificembodiments, at least one ALS inhibitor is applied at a level that wouldsignificantly damage an appropriate control plant. In other embodiments,at least one ALS inhibitor is applied above the recommended label userate for the crop. In still other embodiments, a mixture of ALSinhibitors is applied at a lower rate than the recommended use rate andweeds continue to be selectively controlled. Herbicides that inhibitacetolactate synthase (also known as acetohydroxy acid synthase) and aretherefore useful in the methods of the invention include sulfonylureasas listed in Table 2, including agriculturally suitable salts (e.g.,sodium salts) thereof; sulfonylaminocarbonyltriazolinones as listed inTable 2, including agriculturally suitable salts (e.g., sodium salts)thereof; triazolopyrimidines as listed in Table 2, includingagriculturally suitable salts (e.g., sodium salts) thereof;pyrimidinyloxy(thio)benzoates as listed in Table 2, includingagriculturally suitable salts (e.g., sodium salts) thereof; andimidazolinones as listed in Table 2, including agriculturally suitablesalts (e.g., sodium salts) thereof. In some embodiments, methods of theinvention comprise the use of a sulfonylurea which is notchlorimuron-ethyl, chlorsulfuron, rimsulfuron, thifensulfuron-methyl, ortribenuron-methyl.

Thus, in some embodiments, a transgenic plant of the invention is usedin a method of growing a DP-305423-1 soybean crop by the application ofherbicides to which the plant is tolerant. In this manner, treatmentwith a combination of one of more herbicides which include, but are notlimited to: acetochlor, acifluorfen and its sodium salt, aclonifen,acrolein (2-propenal), alachlor, alloxydim, ametryn, amicarbazone,amidosulfuron, aminopyralid, amitrole, ammonium sulfamate, anilofos,asulam, atrazine, azimsulfuron, beflubutamid, benazolin,benazolin-ethyl, bencarbazone, benfluralin, benfuresate,bensulfuron-methyl, bensulide, bentazone, benzobicyclon, benzofenap,bifenox, bilanafos, bispyribac and its sodium salt, bromacil,bromobutide, bromofenoxim, bromoxynil, bromoxynil octanoate, butachlor,butafenacil, butamifos, butralin, butroxydim, butylate, cafenstrole,carbetamide, carfentrazone-ethyl, catechin, chlomethoxyfen, chloramben,chlorbromuron, chlorflurenol-methyl, chloridazon, chlorimuron-ethyl,chlorotoluron, chlorpropham, chlorsulfuron, chlorthal-dimethyl,chlorthiamid, cinidon-ethyl, cinmethylin, cinosulfuron, clethodim,clodinafop-propargyl, clomazone, clomeprop, clopyralid,clopyralid-olamine, cloransulam-methyl, CUH-35 (2-methoxyethyl2-[[[4-chloro-2-fluoro-5-[(1-methyl-2-propynyl)oxy]phenyl](3-fluorobenzoyl)amino]carbonyl]-1-cyclohexene-1-carboxylate),cumyluron, cyanazine, cycloate, cyclosulfamuron, cycloxydim,cyhalofop-butyl, 2,4-D and its butotyl, butyl, isoctyl and isopropylesters and its dimethylammonium, diolamine and trolamine salts,daimuron, dalapon, dalapon-sodium, dazomet, 2,4-DB and itsdimethylammonium, potassium and sodium salts, desmedipham, desmetryn,dicamba and its diglycolammonium, dimethylammonium, potassium and sodiumsalts, dichlobenil, dichlorprop, diclofop-methyl, diclosulam,difenzoquat metilsulfate, diflufenican, diflufenzopyr, dimefuron,dimepiperate, dimethachlor, dimethametryn, dimethenamid, dimethenamid-P,dimethipin, dimethylarsinic acid and its sodium salt, dinitramine,dinoterb, diphenamid, diquat dibromide, dithiopyr, diuron, DNOC,endothal, EPTC, esprocarb, ethalfluralin, ethametsulfuron-methyl,ethofumesate, ethoxyfen, ethoxysulfuron, etobenzanid, fenoxaprop-ethyl,fenoxaprop-P-ethyl, fentrazamide, fenuron, fenuron-TCA, flamprop-methyl,flamprop-M-isopropyl, flamprop-M-methyl, flazasulfuron, florasulam,fluazifop-butyl, fluazifop-P-butyl, flucarbazone, flucetosulfuron,fluchloralin, flufenacet, flufenpyr, flufenpyr-ethyl, flumetsulam,flumiclorac-pentyl, flumioxazin, fluometuron, fluoroglycofen-ethyl,flupyrsulfuron-methyl and its sodium salt, flurenol, flurenol-butyl,fluridone, flurochloridone, fluroxypyr, flurtamone, fluthiacet-methyl,fomesafen, foramsulfuron, fosamine-ammonium, glufosinate,glufosinate-ammonium, glyphosate and its salts such as ammonium,isopropylammonium, potassium, sodium (including sesquisodium) andtrimesium (alternatively named sulfosate), halosulfuron-methyl,haloxyfop-etotyl, haloxyfop-methyl, hexazinone, HOK-201(N-(2,4-difluorophenyl)-1,5-dihydro-N-(1-methylethyl)-5-oxo-1-[(tetrahydro-2H-pyran-2-yl)methyl]-4H-1,2,4-triazole-4-carboxamide),imazamethabenz-methyl, imazamox, imazapic, imazapyr, imazaquin,imazaquin-ammonium, imazethapyr, imazethapyr-ammonium, imazosulfuron,indanofan, iodosulfuron-methyl, ioxynil, ioxynil octanoate,ioxynil-sodium, isoproturon, isouron, isoxaben, isoxaflutole,isoxachlortole, lactofen, lenacil, linuron, maleic hydrazide, MCPA andits salts (e.g., MCPA-dimethylammonium, MCPA-potassium and MCPA-sodium,esters (e.g., MCPA-2-ethylhexyl, MCPA-butotyl) and thioesters (e.g.,MCPA-thioethyl), MCPB and its salts (e.g., MCPB-sodium) and esters(e.g., MCPB-ethyl), mecoprop, mecoprop-P, mefenacet, mefluidide,mesosulfuron-methyl, mesotrione, metam-sodium, metamifop, metamitron,metazachlor, methabenzthiazuron, methylarsonic acid and its calcium,monoammonium, monosodium and disodium salts, methyldymron, metobenzuron,metobromuron, metolachlor, S-metholachlor, metosulam, metoxuron,metribuzin, metsulfuron-methyl, molinate, monolinuron, naproanilide,napropamide, naptalam, neburon, nicosulfuron, norflurazon, orbencarb,oryzalin, oxadiargyl, oxadiazon, oxasulfuron, oxaziclomefone,oxyfluorfen, paraquat dichloride, pebulate, pelargonic acid,pendimethalin, penoxsulam, pentanochlor, pentoxazone, perfluidone,pethoxyamid, phenmedipham, picloram, picloram-potassium, picolinafen,pinoxaden, piperofos, pretilachlor, primisulfuron-methyl, prodiamine,profoxydim, prometon, prometryn, propachlor, propanil, propaquizafop,propazine, propham, propisochlor, propoxycarbazone, propyzamide,prosulfocarb, prosulfuron, pyraclonil, pyraflufen-ethyl, pyrasulfotole,pyrazogyl, pyrazolynate, pyrazoxyfen, pyrazosulfuron-ethyl,pyribenzoxim, pyributicarb, pyridate, pyriftalid, pyriminobac-methyl,pyrimisulfan, pyrithiobac, pyrithiobac-sodium, pyroxsulam, quinclorac,quinmerac, quinoclamine, quizalofop-ethyl, quizalofop-P-ethyl,quizalofop-P-tefuryl, rimsulfuron, sethoxydim, siduron, simazine,simetryn, sulcotrione, sulfentrazone, sulfometuron-methyl,sulfosulfuron, 2,3,6-TBA, TCA, TCA-sodium, tebutam, tebuthiuron,tefuryltrione, tembotrione, tepraloxydim, terbacil, terbumeton,terbuthylazine, terbutryn, thenylchlor, thiazopyr, thiencarbazone,thifensulfuron-methyl, thiobencarb, tiocarbazil, topramezone,tralkoxydim, tri-allate, triasulfuron, triaziflam, tribenuron-methyl,triclopyr, triclopyr-butotyl, triclopyr-triethylammonium, tridiphane,trietazine, trifloxysulfuron, trifluralin, triflusulfuron-methyl,tritosulfuron and vernolate is disclosed.

Other suitable herbicides and agricultural chemicals are known in theart, such as, for example, those described in WO 2005/041654. Otherherbicides also include bioherbicides such as Alternaria destruensSimmons, Colletotrichum gloeosporiodes (Penz.) Penz. & Sacc., Drechsieramonoceras (MTB-951), Myrothecium verrucaria (Albertini & Schweinitz)Ditmar: Fries, Phytophthora palmivora (Butl.) Butl. and Pucciniathlaspeos Schub. Combinations of various herbicides can result in agreater-than-additive (i.e., synergistic) effect on weeds and/or aless-than-additive effect (i.e. safening) on crops or other desirableplants. Herbicidally effective amounts of any particular herbicide canbe easily determined by one skilled in the art through simpleexperimentation.

Herbicides may be classified into groups and/or subgroups as describedherein above with reference to their mode of action, or they may beclassified into groups and/or subgroups in accordance with theirchemical structure.

Sulfonamide herbicides have as an essential molecular structure featurea sulfonamide moiety (—S(O)₂NH—). As referred to herein, sulfonamideherbicides particularly comprise sulfonylurea herbicides,sulfonylaminocarbonyltriazolinone herbicides and triazolopyrimidineherbicides. In sulfonylurea herbicides the sulfonamide moiety is acomponent in a sulfonylurea bridge (—S(O)₂NHC(O)NH(R)—). In sulfonylureaherbicides the sulfonyl end of the sulfonylurea bridge is connectedeither directly or by way of an oxygen atom or an optionally substitutedamino or methylene group to a typically substituted cyclic or acyclicgroup. At the opposite end of the sulfonylurea bridge, the amino group,which may have a substituent such as methyl (R being CH₃) instead ofhydrogen, is connected to a heterocyclic group, typically a symmetricpyrimidine or triazine ring, having one or two substituents such asmethyl, ethyl, trifluoromethyl, methoxy, ethoxy, methylamino,dimethylamino, ethylamino and the halogens. Insulfonylaminocarbonyltriazolinone herbicides, the sulfonamide moiety isa component of a sulfonylaminocarbonyl bridge (—S(O)₂NHC(O)—). Insulfonylaminocarbonyltriazolinone herbicides the sulfonyl end of thesulfonylaminocarbonyl bridge is typically connected to substitutedphenyl ring. At the opposite end of the sulfonylaminocarbonyl bridge,the carbonyl is connected to the 1-position of a triazolinone ring,which is typically substituted with groups such as alkyl and alkoxy. Intriazolopyrimidine herbicides the sulfonyl end of the sulfonamide moietyis connected to the 2-position of a substituted[1,2,4]triazolopyrimidine ring system and the amino end of thesulfonamide moiety is connected to a substituted aryl, typically phenyl,group or alternatively the amino end of the sulfonamide moiety isconnected to the 2-position of a substituted [1,2,4]triazolopyrimidinering system and the sulfonyl end of the sulfonamide moiety is connectedto a substituted aryl, typically pyridinyl, group.

Representative of the sulfonylurea herbicides useful in the presentinvention are those of the formula:

wherein:

-   -   J is selected from the group consisting of

-   -   J is R¹³SO₂N(CH₃)—;    -   R is H or CH₃;    -   R¹ is F, Cl, Br, NO₂, C₁-C₄ alkyl, C₁-C₄ haloalkyl, C₃-C₄        cycloalkyl, C₂-C₄ haloalkenyl, C₁-C₄ alkoxy, C₁-C₄ haloalkoxy,        C₂-C₄ alkoxyalkoxy, CO₂R¹⁴, C(O)NR¹⁵R¹⁶, SO₂NR¹⁷R¹⁸,        S(O)_(n)R¹⁹, C(O)R²⁰, CH₂CN or L;    -   R² is H, F, Cl, Br, I, CN, CH₃, OCH₃, SCH₃, CF₃ or OCF₂H;    -   R³ is Cl, NO₂, CO₂CH₃, CO₂CH₂CH₃, C(O)CH₃, C(O)CH₂CH₃,        C(O)-cyclopropyl, SO₂N(CH₃)₂, SO₂CH₃, SO₂CH₂CH₃, OCH₃ or        OCH₂CH₃;    -   R⁴ is C₁-C₃ alkyl, C₁-C₂ haloalkyl, C₁-C₂ alkoxy, C₂-C₄        haloalkenyl, F, Cl, Br, NO₂, CO₂R¹⁴, C(O)NR¹⁵R¹⁶, SO₂NR¹⁷R¹⁸,        S(O)_(n)R¹⁹, C(O)R²⁰ or L;    -   R⁵ is H, F, Cl, Br or CH₃;    -   R⁶ is C₁-C₃ alkyl optionally substituted with 0-3 F, 0-1 Cl and        0-1 C₃-C₄ alkoxyacetyloxy, or R⁶ is C₁-C₂ alkoxy, C₂-C₄        haloalkenyl, F, Cl, Br, CO₂R¹⁴, C(O)NR¹⁶R¹⁶, SO₂NR¹⁷R¹⁸,        S(O)_(n)R¹⁹, C(O)R²⁰ or L;    -   R⁷ is H, F, Cl, CH₃ or CF₃;    -   R⁸ is H, C₁-C₃ alkyl or pyridinyl;    -   R⁹ is C₁-C₃ alkyl, C₁-C₂ alkoxy, F, Cl, Br, NO₂, CO₂R¹⁴,        SO₂NR¹⁷R¹⁸, S(O)_(n)R¹⁹, OCF₂H, C(O)R²⁰, C₂-C₄ haloalkenyl or L;    -   R¹⁰ is H, Cl, F, Br, C₁-C₃ alkyl or C₁-C₂ alkoxy;    -   R¹¹ is H, C₁-C₃ alkyl, C₁-C₂ alkoxy, C₂-C₄ haloalkenyl, F, Cl,        Br, CO₂R¹⁴, C(O)NR¹⁶R¹⁶, SO₂NR¹⁷R¹⁸, S(O)_(n)R¹⁹, C(O)R²⁰ or L;    -   R¹² is halogen, C₁-C₄ alkyl or C₁-C₃ alkylsulfonyl;    -   R¹³ is C₁-C₄ alkyl;    -   R¹⁴ is allyl, propargyl or oxetan-3-yl; or R¹⁴ is C₁-C₃ alkyl        optionally substituted by at least one member independently        selected from halogen, C₁-C₂ alkoxy and CN;    -   R¹⁵ is H, C₁-C₃ alkyl or C₁-C₂ alkoxy;    -   R¹⁶ is C₁-C₂ alkyl;    -   R¹⁷ is H, C₁-C₃ alkyl, C₁-C₂ alkoxy, allyl or cyclopropyl;    -   R¹⁸ is H or C₁-C₃ alkyl;    -   R¹⁹ is C₁-C₃ alkyl, C₁-C₃ haloalkyl, allyl or propargyl;    -   R²⁰ is C₁-C₄ alkyl, C₁-C₄ haloalkyl or C₃-C₅ cycloalkyl        optionally substituted by halogen;    -   n is 0, 1 or 2;    -   L is

-   -   L¹ is CH₂, NH or O;    -   R²¹ is H or C₁-C₃ alkyl;    -   X is H, C₁-C₄ alkyl, C₁-C₄ alkoxy, C₁-C₄ haloalkoxy, C₁-C₄        haloalkyl, C₁-C₄ haloalkylthio, C₁-C₄ alkylthio, halogen, C₂-C₅        alkoxyalkyl, C₂-C₅ alkoxyalkoxy, amino, C₁-C₃ alkylamino or        di(C₁-C₃ alkyl)amino;    -   Y is H, C₁-C₄ alkyl, C₁-C₄ alkoxy, C₁-C₄ haloalkoxy, C₁-C₄        alkylthio, C₁-C₄ haloalkylthio, C₂-C₅ alkoxyalkyl, C₂-C₅        alkoxyalkoxy, amino, C₁-C₃ alkylamino, di(C₁-C₃ alkyl)amino,        C₃-C₄ alkenyloxy, C₃-C₄ alkynyloxy, C₂-C₅ alkylthioalkyl, C₂-C₅        alkylsulfinylalkyl, C₂-C₅ alkylsulfonylalkyl, C₁-C₄ haloalkyl,        C₂-C₄ alkynyl, C₃-C₅ cycloalkyl, azido or cyano; and    -   Z is CH or N;

provided that (i) when one or both of X and Y is C₁ haloalkoxy, then Zis CH; and (ii) when X is halogen, then Z is CH and Y is OCH₃, OCH₂CH₃,N(OCH₃)CH₃, NHCH₃, N(CH₃)₂ or OCF₂H. Of note is the present singleliquid herbicide composition comprising one or more sulfonylureas ofFormula I wherein when R⁶ is alkyl, said alkyl is unsubstituted.

Representative of the triazolopyrimidine herbicides contemplated for usein this invention are those of the formula:

wherein:

-   -   R²² and R²³ each independently halogen, nitro, C₁-C₄ alkyl,        C₁-C₄ haloalkyl, C₁-C₄ alkoxy, C₁-C₄ haloalkoxy or C₂-C₃        alkoxycarbonyl;    -   R²⁴ is H, halogen, C₁-C₂ alkyl or C₁-C₂ alkoxy;    -   W is —NHS(O)₂— or —S(O)₂NH—;    -   Y¹ is H, C₁-C₂ alkyl or C₁-C₂ alkoxy;    -   Y² is H, F, Cl, Br, C₁-C₂ alkyl or C₁-C₂ alkoxy;    -   Y³ is H, F or methoxy;    -   Z¹ is CH or N; and    -   Z² is CH or N;        provided that at least one of Y¹ and Y² is other than H.

In the above Markush description of representative triazolopyrimidineherbicides, when W is —NHS(O)₂— the sulfonyl end of the sulfonamidemoiety is connected to the [1,2,4]triazolopyrimidine ring system, andwhen W is —S(O)₂NH— the amino end of the sulfonamide moiety is connectedto the [1,2,4]triazolopyrimidine ring system.

In the above recitations, the term “alkyl”, used either alone or incompound words such as “alkylthio” or “haloalkyl” includesstraight-chain or branched alkyl, such as, methyl, ethyl, n-propyl,i-propyl, or the different butyl isomers. “Cycloalkyl” includes, forexample, cyclopropyl, cyclobutyl and cyclopentyl. “Alkenyl” includesstraight-chain or branched alkenes such as ethenyl, 1-propenyl,2-propenyl, and the different butenyl isomers. “Alkenyl” also includespolyenes such as 1,2-propadienyl and 2,4-butadienyl. “Alkynyl” includesstraight-chain or branched alkynes such as ethynyl, 1-propynyl,2-propynyl and the different butynyl isomers. “Alkynyl” can also includemoieties comprised of multiple triple bonds such as 2,5-hexadiynyl.“Alkoxy” includes, for example, methoxy, ethoxy, n-propyloxy,isopropyloxy and the different butoxy isomers. “Alkoxyalkyl” denotesalkoxy substitution on alkyl. Examples of “alkoxyalkyl” include CH₃OCH₂,CH₃OCH₂CH₂, CH₃CH₂OCH₂, CH₃CH₂CH₂CH₂OCH₂ and CH₃CH₂OCH₂CH₂.“Alkoxyalkoxy” denotes alkoxy substitution on alkoxy. “Alkenyloxy”includes straight-chain or branched alkenyloxy moieties. Examples of“alkenyloxy” include H₂C≡CHCH₂O, (CH₃)CH≡CHCH₂O and CH₂═CHCH₂CH₂O.“Alkynyloxy” includes straight-chain or branched alkynyloxy moieties.Examples of “alkynyloxy” include HCCCH₂O and CH₃CCCH₂O. “Alkylthio”includes branched or straight-chain alkylthio moieties such asmethylthio, ethylthio, and the different propylthio isomers.“Alkylthioalkyl” denotes alkylthio substitution on alkyl. Examples of“alkylthioalkyl” include CH₃SCH₂, CH₃SCH₂CH₂, CH₃CH₂SCH₂,CH₃CH₂CH₂CH₂SCH₂ and CH₃CH₂SCH₂CH₂; “alkylsulfinylalkyl” and“alkylsulfonylalkyl” include the corresponding sulfoxides and sulfones,respectively. Other substituents such as “alkylamino”, “dialkylamino”are defined analogously.

The total number of carbon atoms in a substituent group is indicated bythe “C_(i)-C_(j)” prefix where i and j are numbers from 1 to 5. Forexample, C₁-C₄ alkyl designates methyl through butyl, including thevarious isomers. As further examples, C₂ alkoxyalkyl designates CH₃OCH₂;C₃ alkoxyalkyl designates, for example, CH₃CH(OCH₃), CH₃OCH₂CH₂ orCH₃CH₂OCH₂; and C₄ alkoxyalkyl designates the various isomers of analkyl group substituted with an alkoxy group containing a total of fourcarbon atoms, examples including CH₃CH₂CH₂OCH₂ and CH₃CH₂OCH₂CH₂.

The term “halogen”, either alone or in compound words such as“haloalkyl”, includes fluorine, chlorine, bromine or iodine. Further,when used in compound words such as “haloalkyl”, said alkyl may bepartially or fully substituted with halogen atoms which may be the sameor different. Examples of “haloalkyl” include F₃C, ClCH₂, CF₃CH₂ andCF₃CCl₂. The terms “haloalkoxy”, “haloalkylthio”, and the like, aredefined analogously to the term “haloalkyl”. Examples of “haloalkoxy”include CF₃O, CCl₃CH₂O, HCF₂CH₂CH₂O and CF₃CH₂O. Examples of“haloalkylthio” include CCl₃S, CF₃S, CCl₃CH₂S and ClCH₂CH₂CH₂S.

The following sulfonylurea herbicides illustrate the sulfonylureasuseful for this invention: amidosulfuron(N-[[[[(4,6-dimethoxy-2-pyrimdinyl)amino]carbonyl]amino]-sulfonyl]-N-methylmethanesulfonamide),azimsulfuron(N-[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]-1-methyl-4-(2-methyl-2H-tetrazol-5-yl)-1H-pyrazole-5-sulfonamide),bensulfuron-methyl (methyl2-[[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]methyl]benzoate),chlorimuron-ethyl (ethyl2-[[[[(4-chloro-6-methoxy-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]-benzoate),chlorsulfuron(2-chloro-N-[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino]-carbonyl]benzenesulfonamide),cinosulfuron(N-[[(4,6-dimethoxy-1,3,5-triazin-2-yl)amino]carbonyl]-2-(2-methoxyethoxy)benzenesulfonamide),cyclosulfamuron(N-[[[2-(cyclopropylcarbonyl)phenyl]amino]sulfonyl]-N¹-(4,6-dimethoxypyrimidin-2-yl)urea),ethametsulfuron-methyl (methyl2-[[[[[4-ethoxy-6-(methylamino)-1,3,5-triazin-2-yl]amino]carbonyl]amino]sulfonyl]benzoate),ethoxysulfuron(2-ethoxyphenyl[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]sulfamate),flazasulfuron(N-[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]-3-(trifluoromethyl)-2-pyridinesulfonamide),flucetosulfuron(1-[3-[[[[(4,6-dimethoxy-2-pyrimidinyl)-amino]carbonyl]amino]sulfonyl]-2-pyridinyl]-2-fluoropropylmethoxyacetate), flupyrsulfuron-methyl (methyl2-[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]-amino]sulfonyl]-6-(trifluoromethyl)-3-pyridinecarboxylate),foramsulfuron(2-[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]-4-(formylamino)-N,N-dimethylbenzamide),halosulfuron-methyl (methyl3-chloro-5-[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]-1-methyl-1H-pyrazole-4-carboxylate),imazosulfuron(2-chloro-N-[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]-imidazo[1,2-a]pyridine-3-sulfonamide),iodosulfuron-methyl (methyl4-iodo-2-[[[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino]carbonyl]amino]sulfonyl]benzoate),mesosulfuron-methyl (methyl2-[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]-amino]sulfonyl]-4-[[(methylsulfonyl)amino]methyl]benzoate),metsulfuron-methyl (methyl2-[[[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino]carbonyl]amino]sulfonyl]-benzoate),nicosulfuron(2-[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]amino]-sulfonyl]-N,N-dimethyl-3-pyridinecarboxamide),oxasulfuron (3-oxetanyl2-[[[[(4,6-dimethyl-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]benzoate),primisulfuron-methyl (methyl2-[[[[[4,6-bis(trifluoromethoxy)-2-pyrimidinyl]amino]carbonyl]amino]sulfonyl]benzoate),prosulfuron(N-[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino]carbonyl]-2-(3,3,3-trifluoro-propyl)benzenesulfonamide),pyrazosulfuron-ethyl (ethyl 5-[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]-1-methyl-1H-pyrazole-4-carboxylate),rimsulfuron(N-[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]-3-(ethylsulfonyl)-2-pyridinesulfonamide),sulfometuron-methyl (methyl2-[[[[(4,6-dimethyl-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]benzoate),sulfosulfuron(N-[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]-2-(ethylsulfonyl)imidazo[1,2-a]pyridine-3-sulfonamide),thifensulfuron-methyl (methyl3-[[[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino]carbonyl]amino]sulfonyl]-2-thiophenecarboxylate),triasulfuron(2-(2-chloroethoxy)-N-[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino]carbonyl]benzenesulfonamide),tribenuron-methyl (methyl2-[[[[N-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)-N-methylamino]carbonyl]amino]-sulfonyl]benzoate),trifloxysulfuron(N-[[(4,6-dimethoxy-2-pyrimidinyl)amino]-carbonyl]-3-(2,2,2-trifluoroethoxy)-2-pyridinesulfonamide),triflusulfuron-methyl (methyl2-[[[[[4-dimethylamino)-6-(2,2,2-trifluoroethoxy)-1,3,5-triazin-2-yl]amino]-carbonyl]amino]sulfonyl]-3-methylbenzoate)and tritosulfuron(N-[[[4-methoxy-6-(trifluoromethyl)-1,3,5-triazin-2-yl]amino]carbonyl]-2-(trifluoromethyl)benzene-sulfonamide).

The following triazolopyrimidine herbicides illustrate thetriazolopyrimidines useful for this invention: cloransulam-methyl(methyl3-chloro-2-[[(5-ethoxy-7-fluoro-[1,2,4]triazolo[1,5-c]pyrimidin-2-yl)sulfonyl]amino]benzoate,diclosulam(N-(2,6-dichlorophenyl)-5-ethoxy-7-fluoro[1,2,4]triazolo[1,5-c]pyrimidine-2-sulfonamide,florasulam(N-(2,6-difluorophenyl)-8-fluoro-5-methoxy[1,2,4]triazolo[1,5-c]pyrimidine-2-sulfonamide),flumetsulam(N-(2,6-difluorophenyl)-5-methyl[1,2,4]triazolo[1,5-a]pyrimidine-2-sulfonamide),metosulam(N-(2,6-dichloro-3-methylphenyl)-5,7-dimethoxy[1,2,4]triazolo[1,5-a]pyrimidine-2-sulfonamide),penoxsulam(2-(2,2-difluoroethoxy)-N-(5,8-dimethoxy[1,2,4]triazolo[1,5-c]pyrimidin-2-yl)-6-(trifluoromethyl)benzenesulfonamide)and pyroxsulam(N-(5,7-dimethoxy[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)-2-methoxy-4-(trifluoromethyl)-3-pyridinesulfonamide).

The following sulfonylaminocarbonyltriazolinone herbicides illustratethe sulfonylaminocarbonyltriazolinones useful for this invention:flucarbazone(4,5-dihydro-3-methoxy-4-methyl-5-oxo-N-[[2-(trifluoromethoxy)phenyl]sulfonyl]-1H-1,2,4-triazole-1-carboxamide)and procarbazone (methyl2-[[[(4,5-dihydro-4-methyl-5-oxo-3-propoxy-1H-1,2,4-triazol-1-yl)carbonyl]amino]sulfonyl]benzoate).

Additional herbicides include phenmedipham, triazolinones, and theherbicides disclosed in WO2006/012981, herein incorporated by referencein its entirety.

The methods further comprise applying to the crop and the weeds in afield a sufficient amount of at least one herbicide to which the cropseeds or plants is tolerant, such as, for example, glyphosate, ahydroxyphenylpyruvatedioxygenase inhibitor (e.g., mesotrione orsulcotrione), a phytoene desaturase inhibitor (e.g., diflufenican), apigment synthesis inhibitor, sulfonamide, imidazolinone, bialaphos,phosphinothricin, azafenidin, butafenacil, sulfosate, glufosinate,triazolopyrimidine, pyrimidinyloxy(thio)benzoate, orsulonylaminocarbonyltriazolinone, an acetyl Co-A carboxylase inhibitorsuch as quizalofop-P-ethyl, a synthetic auxin such as quinclorac, or aprotox inhibitor to control the weeds without significantly damaging thecrop plants.

Generally, the effective amount of herbicide applied to the field issufficient to selectively control the weeds without significantlyaffecting the crop. “Weed” as used herein refers to a plant which is notdesirable in a particular area. Conversely, a “crop plant” as usedherein refers to a plant which is desired in a particular area, such as,for example, a soybean plant. Thus, in some embodiments, a weed is anon-crop plant or a non-crop species, while in some embodiments, a weedis a crop species which is sought to be eliminated from a particulararea, such as, for example, an inferior and/or non-transgenic soybeanplant in a field planted with soybean event DP-305423-1, or a maizeplant in a field planted with DP-305423-1. Weeds can be eitherclassified into two major groups: monocots and dicots.

Many plant species can be controlled (i.e., killed or damaged) by theherbicides described herein. Accordingly, the methods of the inventionare useful in controlling these plant species where they are undesirable(i.e., where they are weeds). These plant species include crop plants aswell as species commonly considered weeds, including but not limited tospecies such as: blackgrass (Alopecurus myosuroides), giant foxtail(Setaria faberi), large crabgrass (Digitaria sanguinalis), Surinam grass(Brachiaria decumbens), wild oat (Avena fatua), common cocklebur(Xanthium pensylvanicum), common lambsquarters (Chenopodium album),morning glory (Ipomoea coccinea), pigweed (Amaranthus spp.), velvetleaf(Abutilion theophrasti), common barnyardgrass (Echinochloa crusgalli),bermudagrass (Cynodon dactylon), downy brome (Bromus tectorum),goosegrass (Eleusine indica), green foxtail (Setaria viridis), Italianryegrass (Lolium multiflorum), Johnsongrass (Sorghum halepense), lessercanarygrass (Phalaris minor), windgrass (Apera spica-venti), woolycupgrass (Erichloa villosa), yellow nutsedge (Cyperus esculentus),common chickweed (Stellaria media), common ragweed (Ambrosiaartemisiifolia), Kochia scoparia, horseweed (Conyza canadensis), rigidryegrass (Lolium rigidum), goosegrass (Eleucine indica), hairy fleabane(Conyza bonariensis), buckhorn plantain (Plantago lanceolata), tropicalspiderwort (Commelina benghalensis), field bindweed (Convolvulusarvensis), purple nutsedge (Cyperus rotundus), redvine (Brunnichiaovata), hemp sesbania (Sesbania exaltata), sicklepod (Sennaobtusifolia), Texas blueweed (Helianthus ciliaris), and Devil's claws(Proboscidea louisianica). In other embodiments, the weed comprises aherbicide-resistant ryegrass, for example, a glyphosate resistantryegrass, a paraquat resistant ryegrass, a ACCase-inhibitor resistantryegrass, and a non-selective herbicide resistant ryegrass. In someembodiments, the undesired plants are proximate the crop plants.

As used herein, by “selectively controlled” it is intended that themajority of weeds in an area of cultivation are significantly damaged orkilled, while if crop plants are also present in the field, the majorityof the crop plants are not significantly damaged. Thus, a method isconsidered to selectively control weeds when at least 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or more of the weeds are significantlydamaged or killed, while if crop plants are also present in the field,less than 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% of the cropplants are significantly damaged or killed.

In some embodiments, a soybean DP-305423-1 plant of the invention is notsignificantly damaged by treatment with a particular herbicide appliedto that plant at a dose equivalent to a rate of at least 0.5, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 150, 170, 200, 300,400, 500, 600, 700, 800, 800, 1000, 2000, 3000, 4000, 5000 or more gramsor ounces (1 ounce=29.57 ml) of active ingredient or commercial productor herbicide formulation per acre or per hectare, whereas an appropriatecontrol plant is significantly damaged by the same treatment.

In specific embodiments, an effective amount of an ALS inhibitorherbicide comprises at least about 0.1, 1, 5, 10, 25, 50, 75, 100, 150,200, 250, 300, 350, 400, 450, 500, 600, 700, 750, 800, 850, 900, 950,1000, 2000, 3000, 4000, 5000, or more grams or ounces (1 ounce=29.57 ml)of active ingredient per hectare. In other embodiments, an effectiveamount of an ALS inhibitor comprises at least about 0.1-50, about 25-75,about 50-100, about 100-110, about 110-120, about 120-130, about130-140, about 140-150, about 150-200, about 200-500, about 500-600,about 600-800, about 800-1000, or greater grams or ounces (1 ounce=29.57ml) of active ingredient per hectare. Any ALS inhibitor, for example,those listed in Table 2 can be applied at these levels.

In other embodiments, an effective amount of a sulfonylurea comprises atleast 0.1, 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450,500, 600, 700, 800, 900, 1000, 5000 or more grams or ounces (1ounce=29.57 ml) of active ingredient per hectare. In other embodiments,an effective amount of a sulfonylurea comprises at least about 0.1-50,about 25-75, about 50-100, about 100-110, about 110-120, about 120-130,about 130-140, about 140-150, about 150-160, about 160-170, about170-180, about 190-200, about 200-250, about 250-300, about 300-350,about 350-400, about 400-450, about 450-500, about 500-550, about550-600, about 600-650, about 650-700, about 700-800, about 800-900,about 900-1000, about 1000-2000, or more grams or ounces (1 ounce=29.57ml) of active ingredient per hectare. Representative sulfonylureas thatcan be applied at this level are set forth in Table 2.

In other embodiments, an effective amount of asulfonylaminocarbonyltriazolinones, triazolopyrimidines,pyrimidinyloxy(thio)benzoates, and imidazolinones can comprise at leastabout 0.1, 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100,1150, 1200, 1250, 1300, 1350, 1400, 1500, 1550, 1600, 1650, 1700, 1800,1850, 1900, 1950, 2000, 2500, 3500, 4000, 4500, 5000 or greater grams orounces (1 ounce=29.57 ml) active ingredient per hectare. In otherembodiments, an effective amount of a sulfonyluminocarbonyltriazolines,triazolopyrimidines, pyrimidinyloxy(thio)benzoates, or imidazolinonescomprises at least about 0.1-50, about 25-75, about 50-100, about100-110, about 110-120, about 120-130, about 130-140, about 140-150,about 150-160, about 160-170, about 170-180, about 190-200, about200-250, about 250-300, about 300-350, about 350-400, about 400-450,about 450-500, about 500-550, about 550-600, about 600-650, about650-700, about 700-800, about 800-900, about 900-1000, about 1000-2000,or more grams or ounces (1 ounce=29.57 ml) active ingredient perhectare.

Additional ranges of the effective amounts of herbicides can be found,for example, in various publications from University Extension services.See, for example, Bernards et al. (2006) Guide for Weed Management inNebraska (www.ianrpubs.url.edu/sendIt/ec130); Regher et al. (2005)Chemical Weed Control for Fields Crops, Pastures, Rangeland, andNoncropland, Kansas State University Agricultural Extension Station andCorporate Extension Service; Zollinger et al. (2006) North Dakota WeedControl Guide, North Dakota Extension Service, and the Iowa StateUniversity Extension at www.weeds.iastate.edu, each of which is hereinincorporated by reference.

Herbicides known to inhibit ALS vary in their active ingredient as wellas their chemical formulations. One of skill in the art is familiar withthe determination of the amount of active ingredient and/or acidequivalent present in a particular volume and/or weight of herbicidepreparation.

Rates at which the ALS inhibitor herbicide is applied to the crop, croppart, seed or area of cultivation can be any of the rates disclosedherein. In specific embodiments, the rate for the ALS inhibitorherbicide is about 0.1 to about 5000 g ai/hectare, about 0.5 to about300 g ai/hectare, or about 1 to about 150 g ai/hectare.

Generally, a particular herbicide is applied to a particular field (andany plants growing in it) no more than 1, 2, 3, 4, 5, 6, 7, or 8 times ayear, or no more than 1, 2, 3, 4, or 5 times per growing season.

By “treated with a combination of” or “applying a combination of”herbicides to a crop, area of cultivation or field” it is intended thata particular field, crop or weed is treated with each of the herbicidesand/or chemicals indicated to be part of the combination so that adesired effect is achieved, i.e., so that weeds are selectivelycontrolled while the crop is not significantly damaged. In someembodiments, weeds which are susceptible to each of the herbicidesexhibit damage from treatment with each of the herbicides which isadditive or synergistic. The application of each herbicide and/orchemical may be simultaneous or the applications may be at differenttimes, so long as the desired effect is achieved. Furthermore, theapplication can occur prior to the planting of the crop.

The proportions of herbicides used in the methods of the invention withother herbicidal active ingredients in herbicidal compositions aregenerally in the ratio of 5000:1 to 1:5000, 1000:1 to 1:1000, 100:1 to1:100, 10:1 to 1:10 or 5:1 to 1:5 by weight. The optimum ratios can beeasily determined by those skilled in the art based on the weed controlspectrum desired. Moreover, any combinations of ranges of the variousherbicides disclosed in Table 2 can also be applied in the methods ofthe invention.

Thus, in some embodiments, the invention provides improved methods forselectively controlling weeds in a field wherein the total herbicideapplication may be less than 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%,50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% of that used inother methods. Similarly, in some embodiments, the amount of aparticular herbicide used for selectively controlling weeds in a fieldmay be less than 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%,35%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% of the amount of that particularherbicide that would be used in other methods, i.e., methods notutilizing a plant of the invention.

As used herein, the terms “synergy,” “synergistic,” “synergistically”and derivations thereof, such as in a “synergistic effect” or a“synergistic herbicide combination” or a “synergistic herbicidecomposition” refer to circumstances under which the biological activityof a combination of herbicides, such as at least a first herbicide and asecond herbicide, is greater than the sum of the biological activitiesof the individual herbicides. Synergy, expressed in terms of a “SynergyIndex (SI),” generally can be determined by the method described by Kullet al. Applied Microbiology 9, 538 (1961). See also Colby “CalculatingSynergistic and Antagonistic Responses of Herbicide Combinations,” Weeds15, 20-22 (1967).

In the same manner, in some embodiments, a DP-305423-1 soybean plant ofthe invention shows improved tolerance to a particular formulation of aherbicide active ingredient in comparison to an appropriate controlplant. Herbicides are sold commercially as formulations which typicallyinclude other ingredients in addition to the herbicide activeingredient; these ingredients are often intended to enhance the efficacyof the active ingredient. Such other ingredients can include, forexample, safeners and adjuvants (see, e.g., Green and Foy (2003)“Adjuvants: Tools for Enhancing Herbicide Performance,” in Weed Biologyand Management, ed. Inderjit (Kluwer Academic Publishers, TheNetherlands)). Thus, a DP-305423-1 soybean plant of the invention canshow tolerance to a particular formulation of a herbicide (e.g., aparticular commercially available herbicide product) that is at least1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 22%, 25%,27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%, 100%, 125%,150%, 175%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%,1100%, 1200%, 1300%, 1400%, 1500%, 1600%, 1700%, 1800%, 1900%, or 2000%or more higher than the tolerance of an appropriate control plant thatcontains only a single herbicide tolerance gene which confers toleranceto the same herbicide formulation.

In other methods, a herbicide combination is applied over a DP-305423-1soybean plant, where the herbicide combination produces either anadditive or a synergistic effect for controlling weeds. Suchcombinations of herbicides can allow the application rate to be reduced,a broader spectrum of undesired vegetation to be controlled, improvedcontrol of the undesired vegetation with fewer applications, more rapidonset of the herbicidal activity, or more prolonged herbicidal activity.

An “additive herbicidal composition” has a herbicidal activity that isabout equal to the observed activities of the individual components. A“synergistic herbicidal combination” has a herbicidal activity higherthan what can be expected based on the observed activities of theindividual components when used alone. Accordingly, the presentlydisclosed subject matter provides a synergistic herbicide combination,wherein the degree of weed control of the mixture exceeds the sum ofcontrol of the individual herbicides. In some embodiments, the degree ofweed control of the mixture exceeds the sum of control of the individualherbicides by any statistically significant amount including, forexample, about 1% to 5%, about 5% to about 10%, about 10% to about 20%,about 20% to about 30%, about 30% to 40%, about 40% to about 50%, about50% to about 60%, about 60% to about 70%, about 70% to about 80%, about80% to about 90%, about 90% to about 100%, about 100% to 120% orgreater. Further, a “synergistically effective amount” of a herbiciderefers to the amount of one herbicide necessary to elicit a synergisticeffect in another herbicide present in the herbicide composition. Thus,the term “synergist,” and derivations thereof, refer to a substance thatenhances the activity of an active ingredient (ai), i.e., a substance ina formulation from which a biological effect is obtained, for example, aherbicide.

Plants of the current invention can be crossed with transgenic plantsthat are tolerant to glyphosate, to produce progeny that have toleranceto both glyphosate and inhibitors of ALS.

Weeds that can be difficult to control with glyphosate alone in fieldswhere a crop is grown (such as, for example, a soybean crop) include butare not limited to the following: horseweed (e.g., Conyza canadensis);rigid ryegrass (e.g., Lolium rigidum); goosegrass (e.g., Eleusineindica); Italian ryegrass (e.g., Lolium multiflorum); hairy fleabane(e.g., Conyza bonariensis); buckhorn plantain (e.g., Plantagolanceolata); common ragweed (e.g., Ambrosia artemisifolia); morningglory (e.g., Ipomoea spp.); waterhemp (e.g., Amaranthus spp.); fieldbindweed (e.g., Convolvulus arvensis); yellow nutsedge (e.g., Cyperusesculentus); common lambsquarters (e.g., Chenopodium album); wildbuckwheat (e.g., Polygonium convolvulus); velvetleaf (e.g., Abutilontheophrasti); kochia (e.g., Kochia scoparia); and Asiatic dayflower(e.g., Commelina spp.). In areas where such weeds are found, theDP-305423-1 soybeans are particularly useful in allowing the treatmentof a field (and therefore any crop growing in the field) withcombinations of herbicides that would cause unacceptable damage to cropplants that did not contain both of these polynucleotides. Plants of theinvention that are tolerant to glyphosate and other herbicides such as,for example, sulfonylurea, imidazolinone, triazolopyrimidine,pyrimidinyl(thio)benzoate, and/or sulfonylaminocarbonyltriazolinoneherbicides in addition to being tolerant to at least one other herbicidewith a different mode of action or site of action are particularlyuseful in situations where weeds are tolerant to at least two of thesame herbicides to which the plants are tolerant. In this manner, plantsof the invention make possible improved control of weeds that aretolerant to more than one herbicide.

In the methods of the invention, a herbicide may be formulated andapplied to an area of interest such as, for example, a field or area ofcultivation, in any suitable manner. A herbicide may be applied to afield in any form, such as, for example, in a liquid spray or as solidpowder or granules. In specific embodiments, the herbicide orcombination of herbicides that are employed in the methods comprise atankmix or a premix. A herbicide may also be formulated, for example, asa “homogenous granule blend” produced using blends technology (see,e.g., U.S. Pat. No. 6,022,552, entitled “Uniform Mixtures of PesticideGranules”). The blends technology of U.S. Pat. No. 6,022,552 produces anonsegregating blend (i.e., a “homogenous granule blend”) of formulatedcrop protection chemicals in a dry granule form that enables delivery ofcustomized mixtures designed to solve specific problems. A homogenousgranule blend can be shipped, handled, subsampled, and applied in thesame manner as traditional premix products where multiple activeingredients are formulated into the same granule.

Briefly, a “homogenous granule blend” is prepared by mixing together atleast two extruded formulated granule products. In some embodiments,each granule product comprises a registered formulation containing asingle active ingredient which is, for example, a herbicide, afungicide, and/or an insecticide. The uniformity (homogeneity) of a“homogenous granule blend” can be optimized by controlling the relativesizes and size distributions of the granules used in the blend. Thediameter of extruded granules is controlled by the size of the holes inthe extruder die, and a centrifugal sifting process may be used toobtain a population of extruded granules with a desired lengthdistribution (see, e.g., U.S. Pat. No. 6,270,025).

A homogenous granule blend is considered to be “homogenous” when it canbe subsampled into appropriately sized aliquots and the composition ofeach aliquot will meet the required assay specifications. To demonstratehomogeneity, a large sample of the homogenous granule blend is preparedand is then subsampled into aliquots of greater than the minimumstatistical sample size.

Blends also afford the ability to add other agrochemicals at normal,labeled use rates such as additional herbicides (a 3^(rd)/4^(th)mechanism of action), fungicides, insecticides, plant growth regulatorsand the like thereby saving costs associated with additionalapplications.

Any herbicide formulation applied over the DP-305423-1 soybean plant canbe prepared as a “tank-mix” composition. In such embodiments, eachingredient or a combination of ingredients can be stored separately fromone another. The ingredients can then be mixed with one another prior toapplication. Typically, such mixing occurs shortly before application.In a tank-mix process, each ingredient, before mixing, typically ispresent in water or a suitable organic solvent. For additional guidanceregarding the art of formulation, see T. S. Woods, “The Formulator'sToolbox—Product Forms for Modern Agriculture” Pesticide Chemistry andBioscience, The Food-Environment Challenge, T. Brooks and T. R. Roberts,Eds., Proceedings of the 9th International Congress on PesticideChemistry, The Royal Society of Chemistry, Cambridge, 1999, pp. 120-133.See also U.S. Pat. No. 3,235,361, Col. 6, line 16 through Col. 7, line19 and Examples 10-41; U.S. Pat. No. 3,309,192, Col. 5, line 43 throughCol. 7, line 62 and Examples 8, 12, 15, 39, 41, 52, 53, 58, 132,138-140, 162-164, 166, 167 and 169-182; U.S. Pat. No. 2,891,855, Col. 3,line 66 through Col. 5, line 17 and Examples 1-4; Klingman, Weed Controlas a Science, John Wiley and Sons, Inc., New York, 1961, pp 81-96; andHance et al., Weed Control Handbook, 8th Ed., Blackwell ScientificPublications, Oxford, 1989, each of which is incorporated herein byreference in their entirety.

The methods of the invention further allow for the development ofherbicide combinations to be used with the DP-305423-1 soybean plants.In such methods, the environmental conditions in an area of cultivationare evaluated. Environmental conditions that can be evaluated include,but are not limited to, ground and surface water pollution concerns,intended use of the crop, crop tolerance, soil residuals, weeds presentin area of cultivation, soil texture, pH of soil, amount of organicmatter in soil, application equipment, and tillage practices. Upon theevaluation of the environmental conditions, an effective amount of acombination of herbicides can be applied to the crop, crop part, seed ofthe crop or area of cultivation.

In some embodiments, the herbicide applied to the DP-305423-1 soybeanplants of the invention serves to prevent the initiation of growth ofsusceptible weeds and/or serve to cause damage to weeds that are growingin the area of interest. In some embodiments, the herbicide or herbicidemixture exert these effects on weeds affecting crops that aresubsequently planted in the area of interest (i.e., field or area ofcultivation). In the methods of the invention, the application of theherbicide combination need not occur at the same time. So long as thefield in which the crop is planted contains detectable amounts of thefirst herbicide and the second herbicide is applied at some time duringthe period in which the crop is in the area of cultivation, the crop isconsidered to have been treated with a mixture of herbicides accordingto the invention. Thus, methods of the invention encompass applicationsof herbicide which are “preemergent,” “postemergent,” “preplantincorporation” and/or which involve seed treatment prior to planting.

In one embodiment, methods are provided for coating seeds. The methodscomprise coating a seed with an effective amount of a herbicide or acombination of herbicides (as disclosed elsewhere herein). The seeds canthen be planted in an area of cultivation. Further provided are seedshaving a coating comprising an effective amount of a herbicide or acombination of herbicides (as disclosed elsewhere herein).

“Preemergent” refers to a herbicide which is applied to an area ofinterest (e.g., a field or area of cultivation) before a plant emergesvisibly from the soil. “Postemergent” refers to a herbicide which isapplied to an area after a plant emerges visibly from the soil. In someinstances, the terms “preemergent” and “postemergent” are used withreference to a weed in an area of interest, and in some instances theseterms are used with reference to a crop plant in an area of interest.When used with reference to a weed, these terms may apply to only aparticular type of weed or species of weed that is present or believedto be present in the area of interest. While any herbicide may beapplied in a preemergent and/or postemergent treatment, some herbicidesare known to be more effective in controlling a weed or weeds whenapplied either preemergence or postemergence. For example, rimsulfuronhas both preemergence and postemergence activity, while other herbicideshave predominately preemergence (metolachlor) or postemergence(glyphosate) activity. These properties of particular herbicides areknown in the art and are readily determined by one of skill in the art.Further, one of skill in the art would readily be able to selectappropriate herbicides and application times for use with the transgenicplants of the invention and/or on areas in which transgenic plants ofthe invention are to be planted. “Preplant incorporation” involves theincorporation of compounds into the soil prior to planting.

The time at which a herbicide is applied to an area of interest (and anyplants therein) may be important in optimizing weed control. The time atwhich a herbicide is applied may be determined with reference to thesize of plants and/or the stage of growth and/or development of plantsin the area of interest, e.g., crop plants or weeds growing in the area.The stages of growth and/or development of plants are known in the art.For example, soybean plants normally progress through vegetative growthstages known as V_(E) (emergence), V_(C) (cotyledon), V₁ (unifoliate),and V₂ to V_(N). Soybeans then switch to the reproductive growth phasein response to photoperiod cues; reproductive stages include R₁(beginning bloom), R₂ (full bloom), R₃ (beginning pod), R₄ (full pod),R₅ (beginning seed), R₆ (full seed), R₇ (beginning maturity), and R₈(full maturity). Thus, for example, the time at which a herbicide orother chemical is applied to an area of interest in which plants aregrowing may be the time at which some or all of the plants in aparticular area have reached at least a particular size and/or stage ofgrowth and/or development, or the time at which some or all of theplants in a particular area have not yet reached a particular sizeand/or stage of growth and/or development.

The term “safener” refers to a substance that when added to a herbicideformulation eliminates or reduces the phytotoxic effects of theherbicide to certain crops. One of ordinary skill in the art wouldappreciate that the choice of safener depends, in part, on the cropplant of interest and the particular herbicide or combination ofherbicides included in the synergistic herbicide composition. Exemplarysafeners suitable for use with the presently disclosed herbicidecompositions include, but are not limited to, those disclosed in U.S.Pat. Nos. 4,808,208; 5,502,025; 6,124,240 and U.S. Patent ApplicationPublication Nos. 2006/0148647; 2006/0030485; 2005/0233904; 2005/0049145;2004/0224849; 2004/0224848; 2004/0224844; 2004/0157737; 2004/0018940;2003/0171220; 2003/0130120; 2003/0078167, the disclosures of which areincorporated herein by reference in their entirety. The methods of theinvention can involve the use of herbicides in combination withherbicide safeners such as benoxacor, BCS (1-bromo-4-[(chloromethyl)sulfonyl]benzene), cloquintocet-mexyl, cyometrinil, dichlormid,2-(dichloromethyl)-2-methyl-1,3-dioxolane (MG 191), fenchlorazole-ethyl,fenclorim, flurazole, fluxofenim, furilazole, isoxadifen-ethyl,mefenpyr-diethyl, methoxyphenone((4-methoxy-3-methylphenyl)(3-methylphenyl)-methanone), naphthalicanhydride (1,8-naphthalic anhydride) and oxabetrinil to increase cropsafety. Antidotally effective amounts of the herbicide safeners can beapplied at the same time as the compounds of this invention, or appliedas seed treatments.

Seed treatment is particularly useful for selective weed control,because it physically restricts antidoting to the crop plants. Thereforea particularly useful embodiment of the present invention is a methodfor selectively controlling the growth of weeds in a field comprisingtreating the seed from which the crop is grown with an antidotallyeffective amount of safener and treating the field with an effectiveamount of herbicide to control weeds. Antidotally effective amounts ofsafeners can be easily determined by one skilled in the art throughsimple experimentation. An antidotally effective amount of a safener ispresent where a desired plant is treated with the safener so that theeffect of a herbicide on the plant is decreased in comparison to theeffect of the herbicide on a plant that was not treated with thesafener; generally, an antidotally effective amount of safener preventsdamage or severe damage to the plant treated with the safener. One ofskill in the art is capable of determining whether the use of a safeneris appropriate and determining the dose at which a safener should beadministered to a crop.

In specific embodiments, the combination of safening herbicidescomprises a first ALS inhibitor and a second ALS inhibitor.

Such mixtures provide increased crop tolerance (i.e., a decrease inherbicidal injury). This method allows for increased application ratesof the chemistries post or pre-treatment. Such methods find use forincreased control of unwanted or undesired vegetation. In still otherembodiments, a safening affect is achieved when the DP-305423-1 soybeancrops, crop part, crop seed, weed, or area of cultivation is treatedwith at least one herbicide from the sulfonylurea family of chemistry incombination with at least one herbicide from the imidazolinone family.This method provides increased crop tolerance (i.e., a decrease inherbicidal injury). In specific embodiments, the sulfonylurea comprisesrimsulfuron and the imidazolinone comprises imazethapyr.

As used herein, an “adjuvant” is any material added to a spray solutionor formulation to modify the action of an agricultural chemical or thephysical properties of the spray solution. See, for example, Green andFoy (2003) “Adjuvants: Tools for Enhancing Herbicide Performance,” inWeed Biology and Management, ed. lnderjit (Kluwer Academic Publishers,The Netherlands). Adjuvants can be categorized or subclassified asactivators, acidifiers, buffers, additives, adherents, antiflocculants,antifoamers, defoamers, antifreezes, attractants, basic blends,chelating agents, cleaners, colorants or dyes, compatibility agents,cosolvents, couplers, crop oil concentrates, deposition agents,detergents, dispersants, drift control agents, emulsifiers, evaporationreducers, extenders, fertilizers, foam markers, formulants, inerts,humectants, methylated seed oils, high load COCs, polymers, modifiedvegetable oils, penetrators, repellants, petroleum oil concentrates,preservatives, rainfast agents, retention aids, solubilizers,surfactants, spreaders, stickers, spreader stickers, synergists,thickeners, translocation aids, uv protectants, vegetable oils, waterconditioners, and wetting agents.

In addition, methods of the invention can comprise the use of aherbicide or a mixture of herbicides, as well as, one or more otherinsecticides, fungicides, nematocides, bactericides, acaricides, growthregulators, chemosterilants, semiochemicals, repellents, attractants,pheromones, feeding stimulants or other biologically active compounds orentomopathogenic bacteria, virus, or fungi to form a multi-componentmixture giving an even broader spectrum of agricultural protection.Examples of such agricultural protectants which can be used in methodsof the invention include: insecticides such as abamectin, acephate,acetamiprid, amidoflumet (S-1955), avermectin, azadirachtin,azinphos-methyl, bifenthrin, bifenazate, buprofezin, carbofuran, cartap,chlorfenapyr, chlorfluazuron, chlorpyrifos, chlorpyrifos-methyl,chromafenozide, clothianidin, cyflumetofen, cyfluthrin, beta-cyfluthrin,cyhalothrin, lambda-cyhalothrin, cypermethrin, cyromazine, deltamethrin,diafenthiuron, diazinon, dieldrin, diflubenzuron, dimefluthrin,dimethoate, dinotefuran, diofenolan, emamectin, endosulfan,esfenvalerate, ethiprole, fenothiocarb, fenoxycarb, fenpropathrin,fenvalerate, fipronil, flonicamid, flubendiamide, flucythrinate,tau-fluvalinate, flufenerim (UR-50701), flufenoxuron, fonophos,halofenozide, hexaflumuron, hydramethylnon, imidacloprid, indoxacarb,isofenphos, lufenuron, malathion, metaflumizone, metaldehyde,methamidophos, methidathion, methomyl, methoprene, methoxychlor,metofluthrin, monocrotophos, methoxyfenozide, nitenpyram, nithiazine,novaluron, noviflumuron (XDE-007), oxamyl, parathion, parathion-methyl,permethrin, phorate, phosalone, phosmet, phosphamidon, pirimicarb,profenofos, profluthrin, pymetrozine, pyrafluprole, pyrethrin,pyridalyl, pyriprole, pyriproxyfen, rotenone, ryanodine, spinosad,spirodiclofen, spiromesifen (BSN 2060), spirotetramat, sulprofos,tebufenozide, teflubenzuron, tefluthrin, terbufos, tetrachlorvinphos,thiacloprid, thiamethoxam, thiodicarb, thiosultap-sodium, tralomethrin,triazamate, trichlorfon and triflumuron; fungicides such as acibenzolar,aldimorph, amisulbrom, azaconazole, azoxystrobin, benalaxyl, benomyl,benthiavalicarb, benthiavalicarb-isopropyl, binomial, biphenyl,bitertanol, blasticidin-S, Bordeaux mixture (Tribasic copper sulfate),boscalid/nicobifen, bromuconazole, bupirimate, buthiobate, carboxin,carpropamid, captafol, captan, carbendazim, chloroneb, chlorothalonil,chlozolinate, clotrimazole, copper oxychloride, copper salts such ascopper sulfate and copper hydroxide, cyazofamid, cyflunamid, cymoxanil,cyproconazole, cyprodinil, dichlofluanid, diclocymet, diclomezine,dicloran, diethofencarb, difenoconazole, dimethomorph, dimoxystrobin,diniconazole, diniconazole-M, dinocap, discostrobin, dithianon,dodemorph, dodine, econazole, etaconazole, edifenphos, epoxiconazole,ethaboxam, ethirimol, ethridiazole, famoxadone, fenamidone, fenarimol,fenbuconazole, fencaramid, fenfuram, fenhexamide, fenoxanil,fenpiclonil, fenpropidin, fenpropimorph, fentin acetate, fentinhydroxide, ferbam, ferfurazoate, ferimzone, fluazinam, fludioxonil,flumetover, fluopicolide, fluoxastrobin, fluquinconazole,fluquinconazole, flusilazole, flusulfamide, flutolanil, flutriafol,folpet, fosetyl-aluminum, fuberidazole, furalaxyl, furametapyr,hexaconazole, hymexazole, guazatine, imazalil, imibenconazole,iminoctadine, iodicarb, ipconazole, iprobenfos, iprodione, iprovalicarb,isoconazole, isoprothiolane, kasugamycin, kresoxim-methyl, mancozeb,mandipropamid, maneb, mapanipyrin, mefenoxam, mepronil, metalaxyl,metconazole, methasulfocarb, metiram, metominostrobin/fenominostrobin,mepanipyrim, metrafenone, miconazole, myclobutanil, neo-asozin (ferricmethanearsonate), nuarimol, octhilinone, ofurace, orysastrobin,oxadixyl, oxolinic acid, oxpoconazole, oxycarboxin, paclobutrazol,penconazole, pencycuron, penthiopyrad, perfurazoate, phosphonic acid,phthalide, picobenzamid, picoxystrobin, polyoxin, probenazole,prochloraz, procymidone, propamocarb, propamocarb-hydrochloride,propiconazole, propineb, proquinazid, prothioconazole, pyraclostrobin,pryazophos, pyrifenox, pyrimethanil, pyrifenox, pyrolnitrine,pyroquilon, quinconazole, quinoxyfen, quintozene, silthiofam,simeconazole, spiroxamine, streptomycin, sulfur, tebuconazole,techrazene, tecloftalam, tecnazene, tetraconazole, thiabendazole,thifluzamide, thiophanate, thiophanate-methyl, thiram, tiadinil,tolclofos-methyl, tolyfluanid, triadimefon, triadimenol, triarimol,triazoxide, tridemorph, trimoprhamide tricyclazole, trifloxystrobin,triforine, triticonazole, uniconazole, validamycin, vinclozolin, zineb,ziram, and zoxamide; nematocides such as aldicarb, oxamyl andfenamiphos; bactericides such as streptomycin; acaricides such asamitraz, chinomethionat, chlorobenzilate, cyhexatin, dicofol,dienochlor, etoxazole, fenazaquin, fenbutatin oxide, fenpropathrin,fenpyroximate, hexythiazox, propargite, pyridaben and tebufenpyrad; andbiological agents including entomopathogenic bacteria, such as Bacillusthuringiensis subsp. Aizawai, Bacillus thuringiensis subsp. Kurstaki,and the encapsulated delta-endotoxins of Bacillus thuringiensis (e.g.,Cellcap, MPV, MPVII); entomopathogenic fungi, such as green muscardinefungus; and entomopathogenic virus including baculovirus,nucleopolyhedro virus (NPV) such as HzNPV, AfNPV; and granulosis virus(GV) such as CpGV. The weight ratios of these various mixing partners toother compositions (e.g., herbicides) used in the methods of theinvention typically are between 100:1 and 1:100, or between 30:1 and1:30, between 10:1 and 1:10, or between 4:1 and 1:4.

The present invention also pertains to a composition comprising abiologically effective amount of a herbicide of interest or a mixture ofherbicides, and an effective amount of at least one additionalbiologically active compound or agent and can further comprise at leastone of a surfactant, a solid diluent or a liquid diluent. Examples ofsuch biologically active compounds or agents are: insecticides such asabamectin, acephate, acetamiprid, amidoflumet (S-1955), avermectin,azadirachtin, azinphos-methyl, bifenthrin, binfenazate, buprofezin,carbofuran, chlorfenapyr, chlorfluazuron, chlorpyrifos,chlorpyrifos-methyl, chromafenozide, clothianidin, cyfluthrin,beta-cyfluthrin, cyhalothrin, lambda-cyhalothrin, cypermethrin,cyromazine, deltamethrin, diafenthiuron, diazinon, diflubenzuron,dimethoate, diofenolan, emamectin, endosulfan, esfenvalerate, ethiprole,fenothicarb, fenoxycarb, fenpropathrin, fenvalerate, fipronil,flonicamid, flucythrinate, tau-fluvalinate, flufenerim (UR-50701),flufenoxuron, fonophos, halofenozide, hexaflumuron, imidacloprid,indoxacarb, isofenphos, lufenuron, malathion, metaldehyde,methamidophos, methidathion, methomyl, methoprene, methoxychlor,monocrotophos, methoxyfenozide, nithiazin, novaluron, noviflumuron(XDE-007), oxamyl, parathion, parathion-methyl, permethrin, phorate,phosalone, phosmet, phosphamidon, pirimicarb, profenofos, pymetrozine,pyridalyl, pyriproxyfen, rotenone, spinosad, spiromesifin (BSN 2060),sulprofos, tebufenozide, teflubenzuron, tefluthrin, terbufos,tetrachlorvinphos, thiacloprid, thiamethoxam, thiodicarb,thiosultap-sodium, tralomethrin, trichlorfon and triflumuron; fungicidessuch as acibenzolar, azoxystrobin, benomyl, blasticidin-S, Bordeauxmixture (tribasic copper sulfate), bromuconazole, carpropamid, captafol,captan, carbendazim, chloroneb, chlorothalonil, copper oxychloride,copper salts, cyflufenamid, cymoxanil, cyproconazole, cyprodinil,(S)-3,5-dichloro-N-(3-chloro-1-ethyl-1-methyl-2-oxopropyl)-4-methylbenzamide(RH 7281), diclocymet (S-2900), diclomezine, dicloran, difenoconazole,(S)-3,5-dihydro-5-methyl-2-(methylthio)-5-phenyl-3-(phenyl-amino)-4H-imidazol-4-one(RP 407213), dimethomorph, dimoxystrobin, diniconazole, diniconazole-M,dodine, edifenphos, epoxiconazole, famoxadone, fenamidone, fenarimol,fenbuconazole, fencaramid (SZX0722), fenpiclonil, fenpropidin,fenpropimorph, fentin acetate, fentin hydroxide, fluazinam, fludioxonil,flumetover (RPA 403397), flumorf/flumorlin (SYP-L190), fluoxastrobin(HEC 5725), fluquinconazole, flusilazole, flutolanil, flutriafol,folpet, fosetyl-aluminum, furalaxyl, furametapyr (S-82658),hexaconazole, ipconazole, iprobenfos, iprodione, isoprothiolane,kasugamycin, kresoxim-methyl, mancozeb, maneb, mefenoxam, mepronil,metalaxyl, metconazole, metomino-strobin/fenominostrobin (SSF-126),metrafenone (AC375839), myclobutanil, neo-asozin (ferricmethane-arsonate), nicobifen (BAS 510), orysastrobin, oxadixyl,penconazole, pencycuron, probenazole, prochloraz, propamocarb,propiconazole, proquinazid (DPX-KQ926), prothioconazole (JAU 6476),pyrifenox, pyraclostrobin, pyrimethanil, pyroquilon, quinoxyfen,spiroxamine, sulfur, tebuconazole, tetraconazole, thiabendazole,thifluzamide, thiophanate-methyl, thiram, tiadinil, triadimefon,triadimenol, tricyclazole, trifloxystrobin, triticonazole, validamycinand vinclozolin; nematocides such as aldicarb, oxamyl and fenamiphos;bactericides such as streptomycin; acaricides such as amitraz,chinomethionat, chlorobenzilate, cyhexatin, dicofol, dienochlor,etoxazole, fenazaquin, fenbutatin oxide, fenpropathrin, fenpyroximate,hexythiazox, propargite, pyridaben and tebufenpyrad; and biologicalagents including entomopathogenic bacteria, such as Bacillusthuringiensis subsp. Aizawai, Bacillus thuringiensis subsp. Kurstaki,and the encapsulated delta-endotoxins of Bacillus thuringiensis (e.g.,Cellcap, MPV, MPVII); entomopathogenic fungi, such as green muscardinefungus; and entomopathogenic virus including baculovirus,nucleopolyhedro virus (NPV) such as HzNPV, AfNPV; and granulosis virus(GV) such as CpGV. Methods of the invention may also comprise the use ofplants genetically transformed to express proteins toxic to invertebratepests (such as Bacillus thuringiensis delta-endotoxins). In suchembodiments, the effect of exogenously applied invertebrate pest controlcompounds may be synergistic with the expressed toxin proteins.

General references for these agricultural protectants include ThePesticide Manual, 13th Edition, C. D. S. Tomlin, Ed., British CropProtection Council, Farnham, Surrey, U.K., 2003 and The BioPesticideManual, 2^(nd) Edition, L. G. Copping, Ed., British Crop ProtectionCouncil, Farnham, Surrey, U.K., 2001.

In certain instances, combinations with other invertebrate pest controlcompounds or agents having a similar spectrum of control but a differentmode of action will be particularly advantageous for resistancemanagement. Thus, compositions of the present invention can furthercomprise a biologically effective amount of at least one additionalinvertebrate pest control compound or agent having a similar spectrum ofcontrol but a different mode of action. Contacting a plant geneticallymodified to express a plant protection compound (e.g., protein) or thelocus of the plant with a biologically effective amount of a compound ofthis invention can also provide a broader spectrum of plant protectionand be advantageous for resistance management.

Thus, methods of the invention employ a herbicide or herbicidecombination and may further comprise the use of insecticides and/orfungicides, and/or other agricultural chemicals such as fertilizers. Theuse of such combined treatments of the invention can broaden thespectrum of activity against additional weed species and suppress theproliferation of any resistant biotypes.

Methods of the invention can further comprise the use of plant growthregulators such as aviglycine, N-(phenylmethyl)-1H-purin-6-amine,ethephon, epocholeone, gibberellic acid, gibberellin A₄ and A₇, harpinprotein, mepiquat chloride, prohexadione calcium, prohydrojasmon, sodiumnitrophenolate and trinexapac-methyl, and plant growth modifyingorganisms such as Bacillus cereus strain BP01.

Embodiments of the present invention are further defined in thefollowing Examples. It should be understood that these Examples aregiven by way of illustration only. From the above discussion and theseExamples, one skilled in the art can ascertain the essentialcharacteristics of this invention, and without departing from the spiritand scope thereof, can make various changes and modifications of theembodiments of the invention to adapt it to various usages andconditions. Thus, various modifications of the embodiments of theinvention, in addition to those shown and described herein, will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

Example 1 Genetic Material Used to Produce the DP-305423-1 Event

Soybean (Glycine max) event DP-305423-1 was produced by particleco-bombardment with fragments PHP19340A (FIG. 1; SEQ ID NO:1) andPHP17752A (FIG. 2; SEQ ID NO:2). A summary of the elements and theirposition on the PHP19340A fragment is presented in Table 3 and for thePHP17752A fragment in Table 4. These fragments were obtained by Asc Idigestion from a source plasmid. Fragment PHP19340A was obtained fromplasmid PHP19340 (FIG. 3; SEQ ID NO:3) and fragment PHP17752A wasobtained from plasmid PHP17752 (FIG. 4; SEQ ID NO:4). A summary of theelements and their position on each of the plasmids, PHP19340 andPHP17752, are described in Tables 5 and 6, respectively.

The PHP19340A fragment contains a cassette with a 597 bp fragment of thesoybean microsomal omega-6 desaturase gene 1 (gm-fad2-1) (Heppard etal., 1996). The presence of the gm-fad2-1 fragment in the expressioncassette acts to suppress expression of the endogenous omega-6desaturases, resulting in an increased level of oleic acid and decreasedlevels of palmitic, linoleic, and linolenic acid levels. Upstream of thegm-fad2-1 fragment is the promoter region from the Kunitz trypsininhibitor gene 3 (KTi3) (Jofuku and Goldberg, 1989; Jofuku et al., 1989)regulating expression of the transcript. The KTi3 promoter is highlyactive in soy embryos and 1000-fold less active in leaf tissue (Jofukuand Goldberg, 1989). The 3′ untranslated region of the KTi3 gene (KTi3terminator) (Jofuku and Goldberg, 1989) terminates expression from thiscassette.

The PHP17752A fragment contains a cassette with a modified version ofthe soybean acetolactate synthase gene (gm-hra) encoding the GM-HRAprotein with two amino acid residues modified from the endogenous enzymeand five additional amino acids at the N-terminal region of the proteinderived from the translation of the soybean acetolactate synthase gene5′ untranslated region (Falco and Li, 2003). The gm-hra gene encodes aform of acetolactate synthase, which is tolerant to the sulfonylureaclass of herbicides. The GM-HRA protein is comprised of 656 amino acidsand has a molecular weight of approximately 71 kDa.

The expression of the gm-hra gene is controlled by the 5′ promoterregion of the S-adenosyl-L-methionine synthetase (SAMS) gene fromsoybean (Falco and Li, 2003). This 5′ region consists of a constitutivepromoter and an intron that interrupts the SAMS 5′ untranslated region(Falco and Li, 2003). The terminator for the gm-hra gene is theendogenous soybean acetolactate synthase terminator (gm-als terminator)(Falco and Li, 2003).

TABLE 3 Description of Genetic Elements in the Fragment PHP19340ALocation on DNA Fragment (base pair Genetic Size (base position) Elementpairs) Description  1 to 18 polylinker 18 Region required for cloninggenetic elements region  19 to 2102 KTi3 2084 Promoter region from thesoybean Kunitz trypsin promoter inhibitor gene 3 (Jofuku and Goldberg,1989; Jofuku et al., 1989). 2103 to 2113 polylinker 11 Region requiredfor cloning genetic elements. region 2114 to 2710 gm-fad2-1 597 Fragmentof the soybean microsomal omega-6 fragment desaturase gene (Heppard etal., 1996) 2711 to 2720 polylinker 10 Region required for cloninggenetic elements. region 2721 to 2916 KTi3 196 Terminator region fromthe soybean Kunitz terminator trypsin inhibitor gene 3 (Jofuku andGoldberg, 1989; Jofuku et al., 1989). 2917 to 2924 polylinker 8 Regionrequired for cloning genetic elements region

TABLE 4 Description of Genetic Elements in the Fragment PHP17752ALocation on DNA Fragment (base pair Genetic Size (base position) Elementpairs) Description  1 to 25 polylinker 25 Region required for cloninggenetic elements region 26 to 76 FRT1 51 Flp recombinase recombinationsite (GenBank ID: AY737006.1).  77 to 222 polylinker 145 Region requiredfor cloning genetic elements region 223 to 867 SAMS 645 Promoter portionof the regulatory region of the promoter SAMS gene (Falco and Li, 2003).868 to 926 SAMS 5′- 59 5′ untranslated region of the SAMS gene (FalcoUTR and Li, 2003).  927 to 1517 SAMS intron 591 Intron within the 5′-UTRof the SAMS gene (Falco and Li, 2003). 1518 to 1533 SAMS 5′- 16 5′untranslated region of the SAMS gene (Falco UTR and Li, 2003). 1534 to3504 gm-hra gene 1971 Modified version of the acetolactate synthase genefrom soybean with 15 additional nucleotides on the 5′ end (1534 to 1548)derived from the als 5′ UTR and two nucleotide changes within the codingsequence (Falco and Li, 2003). 3505 to 4156 als terminator 652 Nativeterminator from the soybean acetolactate synthase gene (Falco and Li,2003). 4157 to 4231 polylinker 75 Region required for cloning geneticelements region 4232 to 4282 FRT1 51 Flp recombinase recombination site(GenBank ID: AY737006.1). 4283 to 4396 polylinker 114 Region requiredfor cloning genetic elements region 4397 to 4447 FRT6 51 Modified Flprecombinase recombination site (94% homology to GenBank ID: AY737006.1)4448 to 4512 polylinker 65 Region required for cloning genetic elementsregion

TABLE 5 Description of Genetic Elements of Plasmid PHP19340 Location onKnown Size plasmid (base Genetic (base Region pair position) Elementpairs) Description PHP19340A 2725 to 5438 2924 see Table 3 for elementsand fragment  1 to 210 description of fragment (complement strand)plasmid  211 to 2724 includes 2514 Vector DNA from various sourcesconstruct elements for plasmid construction and below replication. 228to 351 T7 124 Terminator derived from the terminator Enterobacteriaphage T7 genome (GenBank V01146; Dunn and Studier, 1983). (complementstrand)  376 to 1326 Hyg 951 Hygromycin resistance gene from Trypanosomabrucei (GenBank AL671259; Gritz and Davies, 1983). (complement strand)1404 to 1487 T7 84 Promoter derived from the promoter Enterobacteriaphage T7 genome (GenBank V01146; Dunn and Studier, 1983). (complementstrand) 1561 to 1930 Ori 370 Hae II fragment containing bacterial originof replication (colE1 derived) (Tomizawa et al., 1977).

TABLE 6 Description of Genetic Elements of Plasmid PHP17752 Location onKnown Size plasmid (base Genetic (base Region pair position) Elementpairs) Description PHP17752A 2528 to 7026 4512 see Table 4 for elementsand fragment  1 to 13 description of fragment (complement strand)plasmid  14 to 2527 includes 2514 Vector DNA from various sourcesconstruct elements for plasmid construction and below replication.  31to 154 T7 124 Terminator derived from the terminator Enterobacteriaphage T7 genome (GenBank V01146; Dunn and Studier, 1983). (complementstrand)  179 to 1129 Hyg 951 Hygromycin resistance gene from Trypanosomabrucei (GenBank AL671259; Gritz and Davies, 1983). (complement strand)1207 to 1290 T7 84 Promoter derived from the promoter Enterobacteriaphage T7 genome (GenBank V01146; Dunn and Studier, 1983). (complementstrand) 1364 to 1733 Ori 370 Hae II fragment containing bacterial originof replication (colE1 derived) (Tomizawa et al., 1977).

REFERENCES

-   Dunn, J. J. and Studier, F. W. 1983. Complete nucleotide sequence of    bacteriophage T7 DNA and the locations of T7 genetic elements. J.    Mol. Biol 166(4): 477-535.-   Falco, C. S. and Li, Z. 2003. S-adenosyl-L-methionine Synthetase    Promoter and Its Use in Expression of Transgenic Genes in Plants. US    Patent Application: 2003/0226166.-   Gritz, L. and Davies, J. 1983. Plasmid-encoded hygromycin B    resistance: the sequence of hygromycin B phosphotransferase gene and    its expression in E. coli and Saccharomyces cerevisiae. Gene 25:    179-188.-   Heppard, E. P., Kinney, A. J., Stecca, K. L., and Miao, G.-H. 1996.    Developmental and Growth Temperature Regulation of Two Different    Microsomal omega-6 Desaturase Genes in Soybeans. Plant Physiol. 110:    311-319.-   Jofuku, K. D. and Goldberg, R. B. 1989. Kunitz Trypsin Inhibitor    Genes Are Differentially Expressed during the Soybean Life Cycle and    in Transformed Tobacco Plants. Plant Cell 1: 1079-1093.-   Jofuku, K. D. and Schipper, R. D. and Goldberg, R. B. 1989. A    Frameshift Mutation Prevents Kunitz Trypsin Inhibitor mRNA    Accumulation in Soybean Embryos. Plant Cell 1: 427-435.-   Tomizawa, J-I., Ohmori, H., and Bird, R. E. 1977. Origin of    replication of colicin E1 plasmid DNA. Proc. Natl. Acad. Sci. 74    (5): 1865-1869.

Example 2 Method of Transformation and Selection for the Soybean EventDP-305423-1

For transformation of soybean tissue, a linear portion of DNA,containing the gm-fad2-1 gene sequence and the regulatory componentsnecessary for expression, was excised from the plasmid PHP19340 throughthe use of the restriction enzyme Asc I and purified using agarose gelelectrophoresis. A linear portion of DNA, containing the gm-hra genesequences and the regulatory components necessary for expression, wasexcised from the plasmid PHP17752 through the use of the restrictionenzyme Asc I and purified using agarose gel electrophoresis. The linearportion of DNA containing the gm-fad2-1 gene is designated insertPHP19340A and is 2924 bp in size. The linear portion of DNA containingthe gm-hra gene is designated insert PHP17752A and is 4511 bp in size.The only DNA introduced into transformation event DP-305423-1 was theDNA of the inserts described above.

The transgenic plants from event DP-305423-1 were obtained bymicroprojectile bombardment using the Biolistics™ PDS-1000He particlegun manufactured by Bio-Rad, essentially as described by Klein et al.(“High velocity microprojectiles for delivering nucleic acids intoliving cells”, Nature 327:70-73 (1987)). The targets for transformationwere clumps of secondary somatic embryos derived from explants fromsmall, immature soybean seeds. The secondary somatic embryos wereexcised from immature explants after several weeks on a soybean cultureinitiation medium. The embryogenic clumps which were excised from theexplants were transferred to a liquid soybean culture maintenancemedium, and subcultured at regular intervals until prepared forbombardment.

Soybean somatic embryogenic cultures are used in transformationexperiments from 2-4 months after initiation. On the day oftransformation, microscopic gold particles were coated with a mixture ofthe DNA of the two purified fragments, PHP19340A and PHP17752A, andaccelerated into the embryogenic soybean cultures, after which theinsert DNAs were incorporated into some of the cells' chromosomes. OnlyPHP19340A and PHP17752A were used and no additional DNA (e.g., carrierDNA) was incorporated into the transformation process. Afterbombardment, the bombarded soybean tissue was transferred to flasks offresh liquid culture maintenance medium for recovery. After a few days,the liquid culture medium in each flask of bombarded embryogenic soybeanculture was changed to culture maintenance medium supplemented withchlorsulfuron as the selection agent. Individual flasks of tissue inliquid selective medium were kept physically separate during culture,and the majority of the somatic embryogenic clumps within each flaskdied in the liquid selective medium.

After several weeks in the culture maintenance medium supplemented withchlorsulfuron, small islands of healthy, chlorsulfuron-resistant greentissue became visible growing out of pieces of dying somatic embryogenictissue. The resistant embryogenic clumps were excised from theirassociated pieces of dying or dead tissue, and were assigned uniqueidentification codes representing putative transformation events. Theindividual putative events received regular changes to fresh liquidselection medium until the start of the regeneration process.Embryogenic tissue samples were taken for molecular analysis to confirmthe presence of the gm-fad2-1 and gm-hra transgenes by Southernanalysis. Plants were regenerated from tissue derived from each uniqueevent and transferred to the greenhouse for seed production.

Example 3 Southern Analysis of Plants Containing the DP-305423-1 Event

Materials and Methods:

Genomic DNA was extracted from frozen soybean leaf tissue of individualplants of the T4 and T5 generations of DP-305423-1 and of control(variety: Jack) using a standard Urea Extraction Buffer method. GenomicDNA was quantified on a spectrofluorometer using Pico Green® reagent(Molecular Probes, Invitrogen). Approximately 4 μg of DNA per sample wasdigested with Hind III or Nco I. For positive control samples,approximately 3 μg (2 genome copy equivalents) of plasmid PHP19340 orPHP17752 was added to control soybean genomic DNA prior to digestion.Negative control samples consisted of unmodified soybean genomic DNA(variety: Jack). DNA fragments were separated by size using agarose gelelectrophoresis.

Following agarose gel electrophoresis, the separated DNA fragments weredepurinated, denatured, neutralized in situ, and transferred to a nylonmembrane in 20×SSC buffer using the method as described forTURBOBLOTTER™ Rapid Downward Transfer System (Schleicher & Schuell).Following transfer to the membrane, the DNA was bound to the membrane byUV crosslinking.

DNA probes for gm-fad2-1 and gm-hra were labeled with digoxigenin (DIG)by PCR using the PCR DIG Probe Synthesis Kit (Roche).

Labeled probes were hybridized to the target DNA on the nylon membranesfor detection of the specific fragments using DIG Easy Hyb solution(Roche) essentially as described by manufacturer. Post-hybridizationwashes were carried out at high stringency. DIG-labeled probeshybridized to the bound fragments were detected using the CDP-StarChemiluminescent Nucleic Acid Detection System (Roche). Blots wereexposed to X-ray film at room temperature for one or more time points todetect hybridizing fragments.

Summary of Southern Analysis of DP-305423-1:

Schematic maps of plasmids PHP19340 (SEQ ID NO:3) and PHP17752 (SEQ IDNO:4) used as positive controls on these blots are presented in FIGS. 3and 4, respectively. These plasmids were the sources of fragmentsPHP19340A (FIG. 1; SEQ ID NO:1) and PHP17752A (FIG. 2; SEQ ID NO:2). Thefragments were isolated by Asc I digestion of the corresponding sourceplasmid. DP-305423-1 was obtained by particle co-bombardmenttransformation using fragments PHP19340A and PHP17752A.

Genomic DNA isolated from soybean leaf tissue of individual plants ofDP-305423-1 (T5 and T4 generation) and of unmodified control (Jack) wasdigested with Hind III and probed with the gm-fad2-1 gene probe (FIG. 5;Table 7). Approximately 2 μg of genomic DNA was digested and loaded perlane. The gene copy number controls included plasmid PHP19340 andPHP17752 at the indicated approximate gene copy number and 2 μg ofunmodified control DNA. Sizes of the DIG VII molecular weight markersare indicated adjacent to the blot image in kilobase pairs (kb). Adescription of each lane is presented in Table 7.

TABLE 7 Southern Blot Analysis of DP-3Ø5423-1; Hind III Digest,gm-fad2-1 Probe Lane Sample 1 2 copies PHP19340 + Control 2 DIGVII 3Control 4 DP-3Ø5423-1/T8 (T5 generation) 5 DP-3Ø5423-1/T9 (T5generation) 6 DP-3Ø5423-1/T10 (T5 generation) 7 DP-3Ø5423-1/T11 (T5generation) 8 DP-3Ø5423-1/T12 (T5 generation) 9 DP-3Ø5423-1/T13 (T5generation) 10 DP-3Ø5423-1/T14 (T5 generation) 11 DP-3Ø5423-1/T38 (T4generation) 12 DP-3Ø5423-1/T39 (T4 generation) 13 DP-3Ø5423-1/T40 (T4generation) 14 DP-3Ø5423-1/T41 (T4 generation) 15 DP-3Ø5423-1/T42 (T4generation) 16 DP-3Ø5423-1/T43 (T4 generation) 17 DP-3Ø5423-1/T44 (T4generation) 18 Control 19 DIGVII 20 2 copies PHP17752 + Control

Genomic DNA isolated from soybean leaf tissue of individual plants ofDP-305423-1 (T5 and T4 generation) and of unmodified control (Jack) wasdigested with Nco I and probed with the gm-fad2-1 gene probe (FIG. 6;Table 8). Approximately 2 μg of genomic DNA was digested and loaded perlane. The gene copy number controls included plasmid PHP19340 andPHP17752 at the indicated approximate gene copy number and 2 μg ofunmodified control DNA. Sizes of the DIG VII molecular weight markersare indicated adjacent to the blot image in kilobase pairs (kb). Adescription of each lane is presented in Table 8.

TABLE 8 Southern Blot Analysis of DP-3Ø5423-1; Nco I Digest, gm-fad2-1Probe Lane Sample 1 2 copies PHP19340 + Control 2 DIGVII 3 Control 4DP-3Ø5423-1/T8 (T5 generation) 5 DP-3Ø5423-1/T9 (T5 generation) 6DP-3Ø5423-1/T10 (T5 generation) 7 DP-3Ø5423-1/T11 (T5 generation) 8DP-3Ø5423-1/T12 (T5 generation) 9 DP-3Ø5423-1/T13 (T5 generation) 10DP-3Ø5423-1/T14 (T5 generation) 11 DP-3Ø5423-1/T38 (T4 generation) 12DP-3Ø5423-1/T39 (T4 generation) 13 DP-3Ø5423-1/T40 (T4 generation) 14DP-3Ø5423-1/T41 (T4 generation) 15 DP-3Ø5423-1/T42 (T4 generation) 16DP-3Ø5423-1/T43 (T4 generation) 17 DP-3Ø5423-1/T44 (T4 generation) 18Control 19 DIGVII 20 2 copies PHP17752 + Control

Genomic DNA isolated from soybean leaf tissue of individual plants ofDP-305423-1 (T5 and T4 generation) and of unmodified control (Jack) wasdigested with Hind III and probed with the gm-hra gene probe (FIG. 7;Table 9). Approximately 2 μg of genomic DNA was digested and loaded perlane. The gene copy number controls included plasmid PHP19340 andPHP17752 at the indicated approximate gene copy number and 2 μg ofunmodified control DNA. Sizes of the DIG VII molecular weight markersare indicated adjacent to the blot image in kilobase pairs (kb). Adescription of each lane is presented in Table 9.

TABLE 9 Southern Blot Analysis of DP-3Ø5423-1; Hind III Digest, gm-hraProbe Lane Sample 1 2 copies PHP19340 + Control 2 DIGVII 3 Control 4DP-3Ø5423-1/T8 (T5 generation) 5 DP-3Ø5423-1/T9 (T5 generation) 6DP-3Ø5423-1/T10 (T5 generation) 7 DP-3Ø5423-1/T11 (T5 generation) 8DP-3Ø5423-1/T12 (T5 generation) 9 DP-3Ø5423-1/T13 (T5 generation) 10DP-3Ø5423-1/T14 (T5 generation) 11 DP-3Ø5423-1/T38 (T4 generation) 12DP-3Ø5423-1/T39 (T4 generation) 13 DP-3Ø5423-1/T40 (T4 generation) 14DP-3Ø5423-1/T41 (T4 generation) 15 DP-3Ø5423-1/T42 (T4 generation) 16DP-3Ø5423-1/T43 (T4 generation) 17 DP-3Ø5423-1/T44 (T4 generation) 18Control 19 DIGVII 20 2 copies PHP17752 + Control

Genomic DNA isolated from soybean leaf tissue of individual plants ofDP-305423-1 (T5 and T4 generation) and of unmodified control (Jack) wasdigested with Nco I and probed with the gm-hra gene probe. Approximately2 μg of genomic DNA was digested and loaded per lane. The gene copynumber controls included plasmid PHP19340 and PHP17752 at the indicatedapproximate gene copy number and 2 μg of unmodified control DNA. Sizesof the DIG VII molecular weight markers are indicated adjacent to theblot image in kilobase pairs (kb). A description of each lane ispresented in Table 10.

TABLE 10 Southern Blot Analysis of DP-3Ø5423-1; Nco I Digest, gm-hraProbe Lane Sample 1 2 copies PHP19340 + Control 2 DIGVII 3 Control 4DP-3Ø5423-1/T8 (T5 generation) 5 DP-3Ø5423-1/T9 (T5 generation) 6DP-3Ø5423-1/T10 (T5 generation) 7 DP-3Ø5423-1/T11 (T5 generation) 8DP-3Ø5423-1/T12 (T5 generation) 9 DP-3Ø5423-1/T13 (T5 generation) 10DP-3Ø5423-1/T14 (T5 generation) 11 DP-3Ø5423-1/T38 (T4 generation) 12DP-3Ø5423-1/T39 (T4 generation) 13 DP-3Ø5423-1/T40 (T4 generation) 14DP-3Ø5423-1/T41 (T4 generation) 15 DP-3Ø5423-1/T42 (T4 generation) 16DP-3Ø5423-1/T43 (T4 generation) 17 DP-3Ø5423-1/T44 (T4 generation) 18Control 19 DIGVII 20 2 copies PHP17752 + Control

Tables 11 and 12 summarize the results from the Southern blot analysespresented in FIGS. 5 through 8.

TABLE 11 Summary of Expected and Observed Hybridization Fragments onSouthern Blots with the gm-fad2-1 Probe for DP-3Ø5423-1 ExpectedObserved Expected size of Fragment Size in Enzyme Fragment PlasmidDP-3Ø5423-1 Generation Digestion Size¹ (bp) (bp)² (bp) T4 and T5 HindIII 1687   1687 ~8600* (FIG. 5) ~8000* ~2400   1687³ T4 and T5 NcoI >2300 3510 >8600* (FIG. 6) (border) 3 bands >8600 ~7400 ~6100 (faint)~2900  ~900* Footnotes: *Hybridizing band that was also present incontrol samples. This band is determined to be from sequences endogenousto the Jack variety background and is not related to the insertion inDP-3Ø5423-1. ¹Size based on map of fragment PHP19340A in FIG. 2. ²Sizebased on plasmid map of PHP19340 in FIG. 1. ³Size is same as expectedbecause of equivalent migration with plasmid positive control.

TABLE 12 Summary of Expected and Observed Hybridization Fragments onSouthern Blots with the gm-hra Probe for DP-3Ø5423-1 Expected ObservedExpected size of Fragment Size in Enzyme Fragment Plasmid DP-3Ø5423-1Generation Digestion Size¹ (bp) (bp)² (bp) T4 and T5 Hind III   24182418 >8600* (FIG. 7)   1529 1529 ~8600* ~7400* ~5700* ~4600*   2418³~2300* ~2100*   1529³  ~900* T4 and T5 Nco I >3000 4214 >8600* (FIG. 8)(border) 2812 ~8000* >1500 ~6900* (border) ~6100* ~5200* ~4900* ~4500*~3600 ~3200 ~1600* Footnotes: *Hybridizing band that was also present incontrol samples. This band is determined to be from sequences endogenousto the Jack variety background and is not related to the insertion inDP-3Ø5423-1. ¹Size based on map of fragment PHP17752A in FIG. 4. ²Sizebased on plasmid map of PHP17752 in FIG. 3. ³Size is same as expectedbecause of equivalent migration with plasmid positive control.

Hind III digestions were conducted on the genomic DNA samples toevaluate internal fragments and integrity of both PHP19340A (FIG. 1; SEQID NO:1) and PHP17752A (FIG. 2; SEQ ID NO:2) across the T4 and T5generations of DP-305423-1. Nco I was selected to evaluate the copynumber of the gm-fad2-1 and gm-hra elements in DP-305423-1 because ofthe presence of a single restriction enzyme site in each of thetransformation fragments. The single restriction enzyme site would yielda single hybridizing border fragment for each inserted copy of thegm-fad2-1 element and two hybridizing border fragments for each copy ofthe gm-hra gene (Tables 11 and 12, respectively). A border fragment isderived from a restriction site in the insert and the nearestcorresponding restriction site within the adjacent plant genomic DNA.The number of border fragments observed with the gm-fad2-1 and gm-hraprobes would provide an estimate of the number of copies of the elementwithin the DNA insertion of DP-305423-1.

The gm-fad2-1 and gm-hra probes used for Southern analysis were highlyhomologous to sequences in the endogenous soybean genome and thusadditional hybridizing fragments were expected. These hybridizing bandswere determined by their presence in the negative control samples andare indicated in Tables 11 and 12 by an asterisk (*).

To verify the integrity of the 3′ region of the PHP19340A insertion, thegm-fad2-1 was hybridized to the Hind III blot. A single internalfragment of 1687 bp would be expected based on the presence of Hind IIIsites in PHP19340A (Table 11, FIG. 1). The expected band of 1687 bp wasobserved and a second band of approximately 2400 bp was also observed(FIG. 5). In addition, the gm-fad2-1 probe hybridized to two additionalbands in DP-305423-1 that were also present in controls and not due tothe DP-305423-1 insertion (FIG. 5). The 2400 bp band is most likely dueto a partial copy of PHP19340A containing the gm-fad2-1 region. Theseresults indicate the presence of intact copies of PHP19340A as well as apartial copy containing gm-fad2-1 in DP-305423-1. This hybridizationpattern is consistent across the T4 and T5 generations of DP-305423-1analyzed (Table 11).

To determine the number of copies of the gm-fad2-1 element inDP-305423-1, the gm-fad2-1 probe was hybridized to the Nco I blot. Aborder fragment of greater than 2300 bp would be expected for each copygm-fad2-1 (Table 11, FIG. 1). The Nco I blot hybridized to the gm-fad2-1probe showed six hybridizing fragments (FIG. 6). Sizes of these sixhybridizing fragments are given in Table 11. Two additional bands wereobserved and determined to be due to the endogenous soybean genome basedon their presence in negative control samples (Table 11, FIG. 6). Thepresence of six hybridizing fragments indicates that there areapproximately six inserted copies of complete or partial gm-fad2-1elements in the DP-305423-1 genome. This hybridization pattern isconsistent across the T4 and T5 generations of DP-305423-1 analyzed(Table 11), indicating stability of the inserted DNA.

Hybridization of the gm-hra probe to the Hind III blot would verify theintegrity of the inserted PHP17752A fragment as two internal bands of1529 bp and 2418 bp would be expected based on the position of Hind IIIsites on the fragment (Table 12, FIG. 2). These two bands were observedin the hybridization of the Hind III blot with the gm-hra probe (Table12, FIG. 7). Additional hybridizing bands were observed in bothDP-305423-1 and control lanes, indicating that these bands were due toendogenous sequences and not due to the DP-305423-1 insertion (Table 12,FIG. 7). The presence of only the two expected transgenic bands are anindication the PHP17752A fragment inserted intact in the genome. Boththe T4 and T5 generations of DP-305423-1 exhibited the samehybridization pattern (Table 12).

Hybridization of the gm-hra probe to the Nco I blot would verify thenumber of copies of the element in DP-305423-1. Two border fragments,one greater than 1500 bp and a second greater than 3000 bp, would beexpected for each copy of the element based on the position of the Nco Irestriction enzyme site within the gm-hra gene in PHP17752A (Table 12,FIG. 4). Two hybridizing bands, one of approximately 3200 bp and 3600bp, were observed (Table 12, FIG. 8). Additional hybridizing bands wereobserved in both DP-305423-1 and control lanes, indicating that thesebands were due to endogenous sequences and not due to the DP-305423-1insertion (Table 12, FIG. 8). The presence of two transgenic bandsindicates one insertion of the gm-hra gene in the DP-305423-1 genome.This hybridization pattern is consistent across the T4 and T5generations of DP-305423-1 analyzed (Table 12), indicating stability ofthe inserted DNA.

In summary, these restriction enzyme and probe combinations showedconsistent hybridization patterns throughout all individuals analyzedand across the T4 and T5 generations of DP-305423-1. Based on theanalyses reported here, there appear to be approximately six copies ofthe partial or complete gm-fad2-1 element and a single copy of thegm-hra gene in the genome of DP-305423-1. Intact and partial copies ofPHP19340A and a single intact copy of PHP17752A are likely to haveinserted into the genome of DP-305423-1.

Example 4 Confirmation of High Oleic Acid Phenotype by GasChromatography (CG) and Southern Blot Analysis

Prior to planting, remove small seed chips (˜2 mg) from the seedcotyledons using a razor blade. Prepare fatty acid methyl esters (FAMES)from single, matured, soybean seed chips by transesterification usingtrimethylsulfonium hydroxide (TMSH) (Butte, 1983). Place seed chips in a1.5 mL glass gas chromatography vial containing 50 μL of TMSH and 0.5 mLof heptane and incubate for 10 minutes at room temperature whileshaking. Transfer vials to the vial racks on the Gas Chromatograph.Separate and quantify fatty acid methyl esters (3 μL injected fromheptane layer) using a Hewlett-Packard 6890-2 Gas Chromatograph fittedwith an Omegawax 320 fused silica capillary column (Supelco Inc.,Bellfonte, Pa.) and a Flame Ionization Detector (FID). The oventemperature is programmed to hold at 220° C. for 5 min, increase to 240°C. at 20° C./min and hold for an additional minute. A Whatman hydrogengenerator supplies carrier gas and supplies hydrogen for the FID.Retention times are compared to those for methyl esters of commerciallyavailable standards (Nu-Chek Prep, Inc., Elysian, Minn.).

Oil profiles for all seeds are reviewed for elevated oleic acid (18:1)levels as confirmation of the phenotype. The oleic acid level asmeasured by GC is expected to be >70% for DP-305423-1 soybean seeds, and<30% for the control seeds.

Plants are examined by Southern blot analysis to confirm the presence ofthe introduced gm-fad2-1 gene fragment and gm-hra gene in DP-305423-1soybean plants, their absence in control soybean plants, and these dataare correlated with the oleic acid results.

Example 5 Characterization of Insert and Flanking Border Sequence ofSoybean Event DP-305423-1

The insert and flanking border regions of DP-305423-1 genomic DNA wereisolated by PCR amplification and by cosmid cloning. PCR fragments wereeither sequenced directly or cloned into plamid vectors prior tosequencing. Cosmid DNAs were isolated and sequenced.

Partial and intact copies of PHP19340A and a single copy of PHP17752Awere found to be present on four contigs of genomic DNA from theDP-305423-1 event. These four contigs were designated Contig-1 (FIG. 9;SEQ ID NO:5), Contig-2 (FIG. 10; SEQ ID NO:6), Contig-3 (FIG. 11; SEQ IDNO:7) and Contig-4 (FIG. 12; SEQ ID NO:82). Contig-1 has 39,499nucleotides. The 5′ soybean genomic sequence is from nucleotide1-18,651; the insert sequence is from nucleotide 18,652-31579; and the3′ soybean genomic sequence is from nucleotide 31580-39,499. Contig-2has 25,843 nucleotides. The 5′ soybean genomic sequence is fromnucleotide 1-12,163; the insert sequence is from nucleotide12,164-14,494; and the 3′ soybean genomic sequence is from nucleotide14,495-25,843. Contig-3 has 12,465 nucleotides. The 5′ soybean genomicsequence is from nucleotide 1-5750; the insert sequence is fromnucleotide 5751-7813; and the 3′ soybean genomic sequence is fromnucleotide 7814-12,465. Contig-4 has 10,058 nucleotides. The 5′ soybeangenomic sequence is from nucleotide 1-2899; the insert sequence is fromnucleotide 2899-7909; and the 3′ soybean genomic sequence is fromnucleotide 7910-10,058.

Genomic DNA Cloning and Primer Design:

Total genomic DNA from DP-305423-1 soybean was partially digested withrestriction enzymes HindIII and MboI, and cloned into cosmid vectors toconstruct HindIII and MboI cosmid libraries. The cosmid libraries werescreened using a KTi3 promoter fragment as probe. Total three uniqueclones (51-21, 51-9, and H3IIBB19) were identified from HindIII library,and two (mbo30 and mbo22) from MboI library. These five clones wereanalyzed by full-insert sequencing (FIS), a transposon-based sub-cloningmethod to facilitate bi-directional sequencing of a cloned insert fromthe site of the transposition event (MJ Research TGS system; Happa etal., 1999). Sequence analysis showed that 51-21 and mbo30 were overlapclones containing the identical insertion from Contig-1, 51-9 and mbo22were overlap clones containing the identical insertion from Contig-2,and H3IIBB19 contained unique Contig-3. Primers were designed based onthe sequences from the cosmid clones. Genomic PCR was performed toverify the insertions and flanking border regions in DP-305423-1soybean.

Contig-1—Insert and Flanking Genomic Border Regions:

Primers were designed based on the sequence information obtained fromthe cosmid clones 51-21 and mbo30 containing sequence of Contig-1. PCRproducts were amplified from genomic DNA of DP-305423-1 soybean usingprimer pairs A (06-O-1571/06-O-1572, 7103 bp of 5′ insert/genomic borderjunction), B (06-O-1351/06-O-1367, 731 bp of 5′ insert/border junction),C (06-O-1357/06-O-1368, 3226 bp of insert), D (06-O-1357/06-O-1369, 2737bp of insert), E (06-O-1356/06-O-1371, 1800 bp of insert), F(06-O-1360/06-O-1423, 1321 bp of insert), G (06-O-1363/06-O-06-O-1369,1830 bp of insert), H (06-O-1421/06-O-06-O-1367, 2410 b p of insert),and I (06-O-1577/06-O-1578, 2991 bp of 3′ insert/genomic borderjunction) (Table 13), and cloned. PCR products B, C, D, E, F, G, and Hwere directly sequenced to verify the insertion, and A and I wereanalyzed by FIS to verify 5′ and 3′ insert/genomic junctions and theirflanking border regions. No PCR products were amplified when the controlgenomic DNA was used as a template.

For Contig-1, 22452 bp of DP-305423-1 genomic sequence was confirmed(nucleotides 11652-34103 of SEQ ID NO:5), comprising 7000 bp of the 5′flanking border sequence, 2524 bp of the 3′ flanking genomic bordersequence, and 12928 bp of inserted DNA. The insert was found to containone intact PHP19340A fragment, a single, intact PHP17752A fragment, andthree truncated PHP19340A fragments. The first truncated PHP19340Afragment contains a partial KTi3 terminator (180 bp) with 3′ deletion,an intact gm-fad2-1 fragment (597 bp) and an intact KTi3 promoter (2084bp). The second truncated PHP19340A fragment contains a partialgm-fad2-1 fragment (39 bp) with 3′ deletion and an intact KTi3 promoter(2084 bp). The third truncated PHP19340A fragment contains a partialKTi3 promoter (245 bp) with 5′ deletion and a partial gm-fad2-1 fragment(186 bp) with 3′ deletion.

To demonstrate that the identified 5′ and 3′ flanking border sequencesfor Contig-1 are of soybean origin, PCR was performed within the 5′ and3′ flanking border regions (07-O-1889/07-O-1940, 07-O-1892/07-O-1894,respectively) on both DP-305423-1 soybean genomic DNA samples andcontrol samples. The expected PCR products (115 bp for the 5′ flankinggenomic region and 278 bp for the 3′ flanking genomic region) weregenerated from both DP-305423-1 soybean and control samples, indicatingthat the sequences were of soybean genomic origin and not specific toDP-305423-1 soybean. These PCR products were cloned and sequenced. Thesequences from both the DP-305423-1 and control genomic DNA wereidentical.

Contig-2—Insert and Flanking Genomic Border Regions:

Primers were designed based on the sequence information obtained fromthe cosmid clones 51-9 and mbo22 for Contig-2. PCR products wereamplified from genomic DNA of DP-305423-1 soybean using primer pairs J(06-O-1588/06-O-1585, 7642 bp of 5′ insert/genomic border junction), K(06-O-1586/06-O-1403, 2807 bp of 5′ insert/genomic border junction), andL (06-O-1404/06-O-1592, 2845 bp of 3′ insert/genomic border junction)(Table 13), and cloned. PCR products K and L were directly sequenced toverify the insertion and 3′ insert/genomic border junction and itsflanking border region, and J was analyzed by FIS to verify 5′insert/genomic border junctions and its flanking border region. No PCRproducts were amplified when the control genomic DNA was used as atemplate.

For Contig-2, 12667 bp of DP-305423-1 genomic sequence was confirmed(nucleotides 4565-17231 of SEQ ID NO:6), comprising 7599 bp of the 5′flanking genomic border sequence, 2737 bp of the 3′ flanking genomicborder sequence, and 2331 bp of inserted DNA. The insert was found tocontain one truncated PHP19340A fragment, with a partial KTi3 promoter(1511 bp), an intact gm-fad2-1 fragment (597 bp), and an intact KTi3terminator (196 bp).

To demonstrate that the identified 5′ and 3′ flanking border sequencesfor Contig-2 are of soybean origin, PCR was performed within the 5′ and3′ flanking genomic regions (primer pairs 07-O-1895/07-O-1898 and07-O-1905/07-O-1903, respectively) on both DP-305423-1 and controlsoybean genomic DNA samples. The expected PCR products (278 bp for the5′ flanking border region and 271 bp for the 3′ flanking border region)were generated from both DP-305423-1 soybean and control samples,indicating that the sequences were of soybean genomic origin and notspecific to DP-305423-1 soybean. These PCR products were cloned andsequenced. The sequences from both the DP-305423-1 and control genomicDNA were identical.

Contig-3—Insert and Flanking Genomic Border Regions:

Primers were designed based on the sequence information obtained fromthe cosmid clone H3IIB19 for Contig-3. PCR products were amplified ongenomic DNA from DP-305423-1 using primer pairs M (06-O-1669/06-O-1426,2804 bp), N (06-O-1355/06-O-1459, 1335 bp), O (06-O-1569/06-O-1551, 1085bp), and P (05-O-1182/06-O-1672, 2614 bp) (Table 13), and cloned. PCRproducts M, N, O, and P were directly sequenced to verify the insertion,and the 5′ and 3′ insert/genomic junction and their flanking genomicregions. No PCR products were amplified when the control genomic DNA wasused as a template.

For Contig-3, 6789 bp of DP-305423-1 soybean genomic sequence wasconfirmed (nucleotides 3312-10100 of SEQ ID N0:7), comprising 2439 bp ofthe 5′ flanking border sequence, 2287 bp of the 3′ flanking bordersequence, and 2063 bp of inserted DNA. The insert was found to containone truncated PHP19340A fragment with only a partial KTi3 promoter (1550bp), and a 495 bp plasmid backbone fragment. This plasmid backbonefragment was identical to the regions located from 2033 bp to 2527 bp inplasmid PHP19340 and from 1836 bp to 2330 bp in plasmid PHP17752, notincluding the origin of replication (ori). The on in plasmids PHP13940and PHP1772 is located from 1561 to 1930 bp and 1364 to 1733 bp,respectively (Tomizawa et al., 1977).

To demonstrate that the identified 5′ and 3′ flanking border sequencesfor Contig-3 are of soybean origin, PCR was performed within the 5′ and3′ flanking border regions (primer pairs 07-O-1881/07-O-1882 and07-O-1886/07-O-1884, respectively) on both DP-305423-1 soybean genomicDNA samples and control samples. The expected PCR products (262 bp forthe 5′ flanking border region and 280 bp for the 3′ flanking borderregion) were generated from both DP-305423-1 soybean and controlsamples, indicating that the sequences were of soybean genomic originand not specific to DP-305423-1 soybean. These PCR products were clonedand sequenced. The sequences from both the DP-305423-1 and controlgenomic DNA were identical.

Contg-4—Insert and Flanking Genomic Border Regions:

Plasmid libraries and iPCR were used to identify the insert within andthe flanking border regions of Contig-4. Total genomic DNA fromDP-305423-1 soybean was digested with restriction enzymes SpeI and BclI,and run on agarose gels to separate the DNA fragments based on theirmolecular weights. The DNA fragments on agarose gels were transferred tonylon membrane, and hybridized with a gm-fad2-1 probe or a KTi3 promoterprobe. The 2.8 kb and 5.1 kb bands were hybridized with the gm-fad2-1probe after SpeI digestion, and 1.5 kb and 3.3 kb bands were hybridizedwith the KTi3 promoter probe after BclI digestion. All of these bandswere only present in DP-305423-1 plants, but absent in control plants.These four bands were cloned into plasmid vectors to make plasmidlibraries. Positive clones were identified after plasmid libraryscreening with the gm-fad2-1 probe or the KTi3 promoter probe, and weredirectly sequenced. The sequence for Contig-4 is presented in SEQ IDNO:82.

The 2.8 kb band from SpeI digestion and the 3.3 kb band from BclIdigestion were overlapping (referred to as SpeI2.8), containing onetruncated PHP19340A fragment with 159 bp deletion at 3′ end of the KTi3promoter; and the 5.1 band from SpeI digestion and the 1.5 kb band fromBclI digestion were overlapping (referred to as SpeI5.1), containing onetruncated PHP19340A fragment with 649 bp deletion at 3′ end of the KTi3promoter. Since there is a SpeI site within the KTi3 terminator, only148 bp KTi3 terminator sequence was obtained for both SpeI2.8 andSpeI5.1.

Based on the sequence information, primers designed for inverse PCR(iPCR) were used to obtain additional sequence information at the 3′ endof the KTi3 terminator. The iPCR products were either directlysequenced, or cloned and then directly sequenced. Sequence datagenerated from iPCR products with NdeI digestion showed that SpeI2.8contained an intact KTi3 terminator and 35 bp KTi3 terminator in thereverse orientation, and SpeI5.1 contained an intact KTi3 terminator and34 bp KTi3 terminator in the reverse orientation, indicating that thetwo KTi3 terminators of SpeI2.8 and SpeI5.1 arranged as invertedrepeats. Sequence data generated from iPCR products with PacI digestionconfirmed that SpeI2.8 and Spe5.1 are arranged as inverted repeats.

Additional confirmation was done using Southern blot analysis. Totalgenomic DNA from DP-305423-1 and control soybean plants were digestedwith BclI, ClaI and XmnI, run on an agarose gel, transferred to nylonmembrane, and hybridized with the gm-fad2-1 probe. The predicted sizebands were hybridized with the gm-fad2-1 probe: about 3.1 kb band forBclI digestion, about 3.9 kb band for ClaI digestion, and 1.7 kb bandfor XmnI digestion (FIG. 12). Taken together, these results suggest thatthe two KTi3 terminators from SpeI2.8 and SpeI5.1 are arranged ininverted fashion.

For Contig-4, 10058 bp of DP-305423-1 genomic sequence was identified(SEQ ID NO:82), comprising 2899 bp of the 5′ flanking genomic bordersequence, 2149 bp of the 3′ flanking genomic border sequence, and 5010bp of inserted DNA. The insert was believed to contain two truncatedPHP19340A fragments in inverted fashion. The first truncated PHP1930Afragment is located from 2900 to 5163 bp, containing a partial KTi3promoter (1442 bp) with 5′ deletion, an intact gm-fad2-1 fragment (597bp) and an intact KTi3 terminator (196 bp). The second truncatedPHP1930A fragment is located from 5164 to 7919 bp, containing a partialKTi3 promoter (1934 bp) with 5′ deletion, an intact gm-fad2-1 fragment(597 bp) and an intact KTi3 terminator (196 bp) (FIG. 12).

To verify the 5′ and 3′ insert/genomic junctions obtained from plasmidlibraries, PCR was performed on genomic DNA of DP-305423-1 soybeanplants using primer pair Q (HOS-A/HOS-B) to confirm the 5′insert/genomic junction, and primer pair R (HOS-C/HOS-D) to confirm the3′ insert/junction. The expected PCR products were amplified fromDP-305423-1 plants (Table 13), and not from control plants; these PCRproducts were cloned and sequenced. The sequence was confirmed to be thesame as the sequence obtained from plasmid clones.

TABLE 13 Genomic PCR to Confirm the Inserted DNA and Flanking GenomicBorder Regions in DP-305423-1 Soybean PCR Product (size Amplified in bp)Primer Pair PCR System¹ Insert Region A (7103) 06-O-1571/06-O-1572Expand Long 1 5′ flanking Template region and insert B (731)06-O-1351/06-O-1367 High Fidelity 1 5′ flanking region and insert C(3226) 06-O-1357/06-O-1368 Advantage-GC-2 1 Insert D (2737)06-O-1357/06-O1369 Advantage-GC-2 1 Insert E (1800) 06-O-1356/06-O-1371High Fidelity 1 Insert F (1321) 06-O-1360/06-O-1423 Advantage-GC-2 1Insert G (1830) 06-O-1363/06-O-1369 Advantage-GC-2 1 Insert H (2410)06-O-1421/06-O-1367 Advantage-GC-2 1 Insert I (2991) 06-O-1577/06-O-1578Extensor High 1 3′ flanking Fidelity region and insert J (7642)06-O-1588/06-O-1585 Expand Long 2 5′ flanking Template region and insertK (2817) 06-O-1586/06-O-1403 Advantage-GC-2 2 5′ flanking region andinsert L (2845) 06-O-1404/06-O-1592 Advantage-GC-2 2 3′ flanking regionand insert M (2804) 06-O-1669/06-O-1426 Expand Long 3 5′ flankingTemplate region and insert N (1335) 06-O-1355/06-O-1459 High Fidelity 3Insert O (1085) 06-O-1569/06-O-1551 Expand Long 3 3′ flanking Templateregion and insert P (2614) 05-O-1182/06-O-1672 High Fidelity 3 3′flanking region and insert Q (209) HOS-A/HOS-B Taq polymerase 4 5′flanking region and insert R (222) HOS-C/HOS-D Taq polymerase 4 3′flanking region and insert ¹The High Fidelity and Expand Long TemplatePCR systems were purchased from Roche (Mannheim, Germany), theAdvantage-GC-2 PCR system was purchased from Clontech (Palo Alto, CA),the Extensor High Fidelity PCR system was purchased from ABgene (Surrey,UK), and the Taq polymerase was purchased from Fermentas (Hanover, MD).

Example 6 Stability of Contig-1 Insert

The insert in Contig-1 was found to contain one intact PHP19340Afragment (gm-fad2-1 suppression cassette), a single, intact PHP17752Afragment (gm-hra expression cassette), and three truncated PHP19340Afragments. Southern blot analysis conducted on 100 plants from the F2generation of DP-305423-1 identified a single plant that appeared tohave undergone a recombination event that resulted in the removal of theentire gm-hra cassette along with portions of two of the multiple KTi3promoter fragments found in the insertion. A large number of plants fromsegregating generations were analyzed by Polymerase Chain Reaction (PCR)to determine at what frequency this recombination occurs.

Seed was obtained from soybean DP-305423-1 segregating generationsBC1F2, BC2F2, and BC3F2. Each generation consisted of DP-305423-1 ineither the Elite 1 or Elite 2 background. A total of 1060 seeds wereplanted (Table 14).

TABLE 14 Soybean DP-305423-1 Seed Plants Generation Background SeedsPlanted Sampled BC1F2 Elite 1 175 166 BC1F2 Elite 2 150 142 BC2F2 Elite1 65 62 BC2F2 Elite 2 40 36 BC3F2 Elite 1 420 402 BC3F2 Elite 2 210 201

Single leaf punches were collected from plants and genomic DNA wasextracted from the punches utilizing a hot sodium hydroxide and trisextraction method (Truett, G. E., Heeger, P., Mynatt, R. L., Truett, A.A., Walker, J. A. and Warman, M. L. (2000) Preparation of PCR-QualityMouse Genomic DNA with Hot Sodium Hydroxide and Tris (HotSHOT). BioTechniques 29: 52-53.).

Real-time PCR was performed on each DNA sample utilizing an ABI PRISM®7900HT Sequence Detection System and accompanying SDS software (AppliedBiosystems, Inc., Foster City, Calif.). Taq Man® probe and primer setswere designed to detect two insertion target sequences: (1) the 5′junction region between genomic and insert DNA in Contig-1, which wasused as a marker for the gm-fad2-1 suppression cassette (SEQ ID NOs:89,90 and 91), and (2) the region in the insert of Contig-1 spanning theSAMS promoter and gm-hra (SEQ ID NOs:92, 93 and 94). In addition, aTaqMan® probe and primer set for a reference soybean endogenous gene wasused to confirm the presence of amplifiable DNA in each reaction. Theanalysis consisted of quantitative real-time PCR determination ofqualitative positive/negative calls. The extracted DNA was assayed usingoptimized and validated primer and probe concentrations inExtract-N-Amp™ PCR reaction mix containing Rox passive reference dye(Sigma-Aldrich, St. Louis, Mo.). After initial incubations at 50° C. for2 minutes and then at 95° C. for 3 minutes, 40 cycles were conducted asfollows: 95° C. for 15 seconds, 60° C. for 1 minute. Positive ornegative determination for each insertion target was based on comparisonof the C_(T) (threshold cycle) of the insertion target PCR to that ofthe endogenous target.

A total of 1009 plants of three different segregating generations(BC1F2, BC2F2 and BC3F2) and two different backgrounds (Elite 1 andElite 2) were analyzed by qualitative real-time PCR for the Contig-1 5′junction and the SAMS Promoter::gm-hra targets. Each reaction containedamplifiable DNA based on the endogenous gene control. Of the 1009 plantsin the six segregating populations, 745 were positive and 264 werenegative for both PCR assays. No plants were identified in which the PCRresults were positive for one target and negative for the other.Consequently, in this sample group of 1009 plants, no recombinationwithin the Contig-1 insertion was detected that selectively removed theSAMS Promoter::gm-hra cassette. A summary of the results is given inTable 15.

TABLE 15 Results of Real-time Qualitative PCR Analysis by Generation andBackground Contig-1 5′ SAMS Junction PCR Promoter::gm- Back- Results hraPCR Results Total Generation ground Positive Negative Positive NegativePlants BC1F2 Elite 1 125 41 125 41 166 Elite 2 108 34 108 34 142 BC2F2Elite 1 39 23 39 23 62 Elite 2 27 9 27 9 36 BC3F2 Elite 1 297 105 297105 402 Elite 2 149 52 149 52 201 Total 745 264 745 264 1009

Example 7 Fatty Acid Levels in Soybean Grain

Levels of 25 fatty acids were measured in DP-305423-1 and controlsoybean grain. Levels of ten fatty acids were below the lower limit ofquantitation (LLOQ) for the assay: caprylic acid (C8:0), capric acid(C10:0), lauric acid (C12:0), myristoleic acid (C14:1), pentadecanoicacid (C15:0), pentadecenoic acid (C15:1), γ-linolenic acid (C18:3),eicosatrienoic acid (C20:3), arachidonic acid (C20:4), and erucic acid(C22:1). Therefore, no statistical analyses were conducted on thesefatty acids and data are not shown. Results of the analysis for the 15remaining fatty acid are presented in Table 16.

The mean values for oleic acid (C18:1) and linoleic acid (C18:2) felloutside the tolerance intervals and/or the combined literature rangesfor conventional soybean varieties. As expected, the mean level of theoleic acid in DP-305423-1 soybean was above the upper range of both thestatistical tolerance interval for the reference soybean lines andliterature range for conventional soybean varieties. The mean level ofthe oleic acid in DP-305423-1 soybean was statistically significantlydifferent from that of the control near isoline soybean (adjustedP-value<0.05). The mean level of linoleic acid (C18:2) in DP-305423-1soybean was below the lower range of the statistical tolerance intervalfor the reference soybean lines and literature range for conventionalsoybean varieties. It was also statistically significantly differentfrom that of the control near isoline soybean (adjusted P-value<0.05).The increase in the oleic acid content and the decrease in linoleic acidcontent in DP-305423-1 soybean are intended effects achieved throughintroduction of the gm-fad2-1 gene fragment. These changes have beenreported previously for transgenic high oleic soybean (OECD identifierDD-Ø26ØØ5-3, AGBIOS database) generated via introduction of the FAD2-1gene (Kinney and Knowlton, 1997; Glancey et al., 1998; Knowlton, 1999).

Though being within the literature ranges and/or statistical toleranceintervals, the mean values for palimitic acid (C16:0) and linolenic acid(C18:3) were statistically significantly different (lower) inDP-305423-1 soybean as compared to the control near isoline (adjustedP-value<0.05). Linolenic acid is produced directly from conversion oflinoleic acid and therefore the decrease in the linoleic acid contentwas expected to affect the linolenic acid content in DP-305423-1soybean. The decrease in both palmitic acid and linolenic acid contenthas been reported previously for transgenic high oleic soybean (OECDidentifier DD-Ø26ØØ5-3, AGBIOS database) generated via introduction ofthe FAD2-1 gene (Kinney and Knowlton, 1997; Glancey et al., 1998;Knowlton, 1999).

The (9,15) isomer of linoleic acid (cis-9, cis-15-octadecadienoic acid)was detected in DP-305423-1 soybean at the mean concentration of 0.341%of the total fatty acids, while the conventional reference varieties didnot contain measurable concentrations of this analyte. This was anexpected finding, as the 9,15-linoleic acid isomer had been previouslyseen in high oleic soybean oil at less than 1% of the total fatty acidcontent (Kinney and Knowlton, 1997). This isomer is also found, atconcentrations ranging from 0.02% to 5.4% of the total fatty acids, inmany edible sources of fat including butterfat, cheese, beef and muttontallow, partially hydrogenated vegetable oils, human milk and mango pulp(Kinney and Knowlton, 1997, and references therein). The 9,15-linoleicacid isomer is likely a result of the activity of the fatty aciddesaturase encoded by the FAD3 gene that normally inserts a d-15 doublebond into 9,12-linoleic acid to produce 9,12,15-linolenic acid. In theDP-305423-1 soybean, the 9,12-linoleic acid content is significantlyreduced (Table 16) so that the FAD3-encoded desaturase probably createsa small amount of the 9,15-linoleic acid isomer by desaturating theabundant 9-oleic acid substrate at the d-15 position. This view issupported by the results of crossing high oleic soybeans (OECDidentifier DD-Ø26ØØ5-3, AGBIOS database) with soybeans containing asilenced FAD3 gene. In the resulting progeny the 9,15-linoleic acidisomer is either reduced or eliminated (Kinney and Knowlton, 1997).

The mean values of two minor fatty acids, heptadecanoic acid (C17:0) andheptadecenoic acid (C17:1), in DP-305423-1 soybean were above the upperrange of the statistical tolerance intervals and literature ranges forconventional soybean varieties. Mean values for C17:0 and C17:1 werestatistically significantly different from those of control near isolinesoybean. However, levels of heptadecanoic and heptadecenoic acid are ingeneral still very low; each represents less than 1.2% of the totalfatty acid content in DP-305423-1 soybean.

The detected increase in heptadecanoic acid (C17:0) and heptadecenoicacid (C17:1), in DP-305423-1 soybean is not unexpected, as expression ofthe GM-HRA protein likely results in a slight shift in availability ofthe GM-HRA enzyme substrates, pyruvate and 2-ketobutyrate. These twocompounds are also substrates for the enzyme complex that initiates oilbiosynthesis.

The mean values for myristic acid (C14:0), palmitoleic acid (C16:1),stearic acid (C18:0), arachidic acid (C20:0), eicosenoic acid (C20:1),behenic acid (C22:0) and lignoceric acid (C24:0) for DP-305423-1 soybeanwere within the statistical tolerance intervals and/or the combinedliterature ranges for these fatty acids in different soybean varieties.With exception of the behenic acid, the mean values for these fattyacids were statistically significantly different either above(palmitoleic, arachidic, eicosenoic, and lignoceric acids) or below(myristic and stearic acids) those in the control near isoline.Myristic, palmitoleic, arachidic, eicosenoic, behenic, and lignocericacids are minor fatty acids, each comprising 0.05-0.5% of the totalfatty acids in DP-305423-1 soybean; stearic acid comprises less then4.5% in DP-305423-1 soybean. These fatty acids are common constituentsof vegetable oils and common foodstuffs and are present at levelssimilar to those observed in DP-305423-1 soybean (USDA NutritionDatabase, Release 19).

Eicosadienoic acid (C20:2) was undetectable in DP-305423-1 soybean.Similarly, reference soybean varieties also lacked measurableconcentrations of this fatty acid. A very low level of the eicosadienoicacid was detectable in the control near isoline soybean; however, thisdifference with DP-305423-1 soybean was not statistically significant(adjusted P-value>0.05).

TABLE 16 Major Fatty Acids in Soybean Grain Control Combined Fatty Acid(Null 305423 Tolerance Literature (% Total) Segregant) Soybean Interval¹Ranges² Myristic Acid Mean³ 0.0742 0.0451    0-0.174 0.0710-0.238 (C14:0) Range⁴ 0.0676-0.0807 0.0419-0.0522 Adjusted P- 0.0007⁷ value⁵P-value⁶ 0.0001 Palmitic Acid Mean 10.3 6.28 2.93-19.6 7.00-15.8 (C16:0)Range 9.77-10.7 5.71-7.27 Adjusted P- 0.0007⁷ value P-value 0.0001Palmitoleic Mean 0.0860 0.0946 0.0110-0.177  0.0860-0.194  Acid (C16:1)Range 0.0751-0.0948 0.0835-0.105  Adjusted P- 0.0248⁷ value P-value0.0053 Heptadecanoic Mean 0.113 0.798 0.0722-0.131  0.0850-0.146  Acid(C17:0) Range 0.0993-0.127  0.703-0.890 Adjusted P- 0.0007⁷ valueP-value 0.0001 Heptadecenoic Mean 0.0614 1.19 0.0351-0.07320.0730-0.0870 Acid (C17:1) Range 0.0513-0.0762 1.01-1.51 Adjusted P-0.0007⁷ value P-value 0.0001 Stearic Acid Mean 4.98 4.36 0.852-8.34 2.00-5.88 (C18:0) Range 4.36-5.89 3.90-5.01 Adjusted P- 0.0007⁷ valueP-value 0.0001 Oleic Acid Mean 21.1 76.5 11.3-32.6 14.3-34.0 (C18:1)Range 18.0-24.1 68.7-79.4 Adjusted P- 0.0007⁷ value P-value 0.0001Linoleic Acid Mean 52.5 3.62 41.7-64.3 42.3-60.0 (C18:2) Range 50.2-54.31.53-8.98 Adjusted P- 0.0007⁷ value P-value 0.0001 Linoleic Acid Mean0.247 0.341 NA⁸ NR⁹ (C18:2) Range    0-0.532 0.143-0.456 Isomer (9,15)Adjusted P- 0.1787 value P-value 0.0699 Linolenic Acid Mean 9.35 5.391.15-14.7 2.00-12.5 (C18:3) Range 7.83-11.2 4.03-7.32 Adjusted P-0.0007⁷ value P-value 0.0001 Arachidic Acid Mean 0.396 0.450 0.103-0.619  0-1.00 (C20:0) Range 0.348-0.479 0.393-0.528 Adjusted P- 0.0007⁷ valueP-value 0.0001 Eicosenoic Mean 0.170 0.347 0.0549-0.319  0.140-0.350Acid (C20:1) Range 0.135-0.201 0.290-0.394 Adjusted P- 0.0007⁷ valueP-value 0.0001 Eicosadienoic Mean 0.0225 0 NA⁸ 0.0770-0.245  Acid(C20:2) Range    0-0.0502 0-0 Adjusted P- 0.0928 value P-value 0.0298Behenic Acid Mean 0.414 0.427 0.188-0.458 0.277-0.595 (C22:0) Range0.349-0.566 0.382-0.546 Adjusted P- 0.5468 value P-value 0.3779Lignoceric Mean 0.114 0.143    0-0.310 NR⁹ Acid (C24:0) Range0.0845-0.139  0.115-0.173 Adjusted P- 0.0017⁷ value P-value 0.0003¹Negative tolerance limits have been set to zero. ²Literature ranges aretaken from published literature for soybeans (OECD, 2001; ILSI 2004).³Least Square Mean ⁴Range denotes the lowest and highest individualvalue across locations. ⁵False Discovery Rate (FDR) adjusted P-value⁶Non-adjusted P-value ⁷Statistically significant difference; adjustedP-value <0.05 ⁸Statistical analysis was not available (NA), due to lackof measurable concentrations detected for this analyte. ⁹Analyte rangeswere not reported (NR) in the published literature references.

The article “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one or more element.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed:
 1. A soybean plant or seed comprising a polynucleotidehaving 99% identity to nucleotides 2899-7909 of SEQ ID NO: 82 andfurther comprising SEQ ID NO: 83 and SEQ ID NO:
 84. 2. The soybean plantor seed of claim 1, wherein the seed has an oleic acid content greaterthan 70% and a progeny seed produced by the plant has an oleic acidcontent greater than 70%.
 3. The soybean plant or seed of claim 1,wherein the soybean plant or seed further comprises a polynucleotidehaving 99% identity to nucleotides 18,652-31,579 of SEQ ID NO:
 5. 4. Thesoybean plant or seed of claim 3, wherein the soybean plant or seedfurther comprises a polynucleotide having 99% identity to nucleotides12,164-14,494 of SEQ ID NO:6.
 5. The soybean plant or seed of claim 4,wherein the soybean plant or seed further comprises a polynucleotidehaving 99% identity to nucleotides 5751-7813 of SEQ ID NO:7.
 6. Thesoybean plant or seed of claim 5, wherein the seed has an oleic acidcontent greater than 70% and a progeny seed produced by the plant has anoleic acid content greater than 70%.
 7. The soybean plant or seed ofclaim 6, wherein the plant is tolerant to an ALS-inhibitor herbicide anda plant grown from the seed is tolerant to an ALS-inhibitor herbicide.8. The soybean plant or seed of claim 3, wherein the plant is tolerantto an ALS-inhibitor herbicide and a plant grown from the seed istolerant to an ALS-inhibitor herbicide.
 9. The soybean plant or seed ofclaim 8, wherein the seed has an oleic acid content greater than 70% anda progeny seed produced by the plant has an oleic acid content greaterthan 70%.
 10. The soybean plant or seed of claim 3, wherein the seed hasan oleic acid content greater than 70% and a progeny seed produced bythe plant has an oleic acid content greater than 70%.
 11. The soybeanplant or seed of claim 1, wherein the seed has an oleic acid content of68.7% to 79.4% and a progeny seed produced by the plant has an oleicacid content 68.7% to 79.4%.
 12. The soybean plant or seed of claim 5,wherein the seed has an oleic acid content of 68.7% to 79.4% and aprogeny seed produced by the plant has an oleic acid content 68.7% to79.4%.
 13. A method of producing a plant comprising high oleic seed, themethod comprising breeding the plant of claim 1, and selecting progenyby analyzing for progeny that comprises the polynucleotide having 99%identity to nucleotides 2899-7909 of SEQ ID NO: 82, wherein the progenycomprises SEQ ID NO: 83 and SEQ ID NO: 84 and further comprises higholeic seed.
 14. A soybean plant or seed comprising a polynucleotidehaving 99% identity to nucleotides 18,652-31,579 of SEQ ID NO: 5 andfurther comprising SEQ ID NO: 8 and SEQ ID NO:
 9. 15. The soybean plantor seed of claim 14, wherein the plant is tolerant to an ALS-inhibitorherbicide and a plant grown from the seed is tolerant to anALS-inhibitor herbicide.
 16. The soybean plant or seed of claim 14,wherein the soybean plant or seed further comprises a polynucleotidehaving 99% identity to nucleotides 2899-7909 of SEQ ID NO:
 82. 17. Thesoybean plant or seed of claim 16, wherein the seed has an oleic acidcontent greater than 70% and a progeny seed produced by the plant has anoleic acid content greater than 70%.
 18. A method for controlling weedsin an area of cultivation, the method comprising applying an effectiveamount of an ALS inhibitor to the area of cultivation comprising thesoybean plant of claim
 14. 19. A method of producing an ALS inhibitortolerant plant, the method comprising breeding the plant of claim 14,and selecting progeny by analyzing for progeny that comprise apolynucleotide comprising nucleotides 18,652-31,579 of SEQ ID NO: 5wherein the progeny comprises SEQ ID NO: 8 and SEQ ID NO: 9 and furthercomprises tolerance to an ALS inhibitor.
 20. A method for controllingweeds in an area of cultivation, the method comprising applying aneffective amount of at least one sulfonylurea herbicide and at least oneimidazolinone herbicide to the area of cultivation comprising thesoybean plant of claim 15.